ATPase Regulation in the Maltose Transporter

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1 Western University Electronic Thesis and Dissertation Repository February 2012 ATPase Regulation in the Maltose Transporter Alister D. Gould The University of Western Ontario Supervisor Dr. Brian Shilton The University of Western Ontario Graduate Program in Biochemistry A thesis submitted in partial fulfillment of the requirements for the degree in Doctor of Philosophy Alister D. Gould 2011 Follow this and additional works at: Part of the Biochemistry Commons, and the Structural Biology Commons Recommended Citation Gould, Alister D., "ATPase Regulation in the Maltose Transporter" (2011). Electronic Thesis and Dissertation Repository This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact tadam@uwo.ca, wlswadmin@uwo.ca.

2 ATP AS E R EGU LA T IO N IN THE M A LT OS E TR ANS P OR TER S p i n e Ti t l e: M al FGK 2 ATP as e R e gu l at i o n Th es i s fo rm at : In t e gr at ed -Art i cl e b y Al i s t er D. Go u l d Grad u at e P ro g ram i n Bi o ch em i s t r y Th es i s s ub m i t t ed i n p art i al fu l fi l l m en t o f t h e req u i r em en t s fo r t h e d e gre e o f Do ct o r o f P h i l o s op h y Th e S ch o o l o f Gr ad u at e an d P o s t d o ct o ral S t u d i es Th e Un i v ers i t y o f W es t er n On t ari o Lo n d o n, On t ari o, C an ad a Al i s t er D. Go u l d

3 THE UN IVER S ITY OF W ES TER N ONTAR IO S C HOO L O F GR AD U ATE AND P OS TDOC T OR A L S TUD IES CERTIFICATE OF EXAMINATION Sup e r vi so r _ D r. B r ia n Shilto n Sup e r vi so r y Co m mitte e _ D r. Sta n D u n n _ D r. J a me s Cho y E xa mine r s _ D r. M e ga n D a ve y _ D r. D a vid H e inr ic hs _ D r. V o ula K a ne li s _ D r. G a r y S ha w Th e t h es i s b y Alister D. Gould en t i t l ed : ATPase Regulation in the Maltose Transporter i s accep t ed i n p art i al fu l fi l m en t o f t h e req u i rem en t s fo r t h e d e g r ee o f Do ct o r o f P h i l o s op h y Dat e _ C h ai r o f t h e Th es i s Ex am i n at i o n Bo ard ii

4 Abstract Th i s t h es i s i n v es t i gat es t h e m ech an i s m o f act i v i t y-co up l i n g i n t h e m al t o s e t ran s p o rt er o f Escherichia coli (M al FG K 2 ); t h e wa y ATP h yd ro l ys i s i s p rev en t ed i n t h e a b s ence o f m al t o s e, an d t h en en ab l ed t o d ri v e m al t o s e t ran s p o rt. Li k e o t h er ATP b i n d i n g c as s et t e i m p o rt ers, M al FGK 2 req u i r es s ub s t r at e t o b e p res en t ed b y a p eri p h er al s ub s t rat e-b i n d i n g p ro t ei n, i n t h i s cas e t h e m al t o s e b i n d i n g p ro t e i n (M BP ). M BP p r ed o m i n an t l y ad op t s an op en res t i n g s t at e, b u t u n d er go es a ro t at i o n o f i t s t wo d o m ai n s t o a cl o s e d s t at e aft e r m al t o s e b i n d i n g. In t h e cl o s ed s t at e M BP i s ab l e t o act i v at e M al F GK 2 t o s t i m u l at e ATP h yd ro l ys i s an d m al t o s e t ran s p o rt. En gi n e ered m u t an t s o f M BP h av e b een u s ed t o t es t t he res p o n s i v en es s o f t h e t ran s p o rt e r t o ch an ges i n t h e co n fo r m at i o n al s t at e o f i t s b i n d in g p ro t ei n. C a refu l an al ys i s o f ATP as e s t i m u l u s b y wi l d t yp e M BP i n d i cat es t h at t h e op e n st at e o f M BP i s ab l e t o b i n d t h e t ran s p o rt er an d p ro m o t e ATP h yd ro l ys i s i n t h e ab s en ce o f m a l t o se. An o t h er m u t an t M BP, ab l e t o ad op t t h e cl o s ed s t at e i n t h e p res en ce o f ei t h e r s u cro s e o r m al t o s e, d em o n s t rat es t h at ATP h yd ro l ys i s i s act i v at ed b y i n t er act i o n s b et ween t h e t r an s p o rt e r a n d b i n d i n g p ro t ei n, an d n o t b et ween t h e t ran s p o rt er an d s ub s t rat e. Fu rt h er, u s i n g M BP m u t an t s i n co rp o rat i n g i n t ro d u ced d i s u l fi d e b o n d s an d i n t er-d o m ai n c ro s s -l i n k ers we s h o w t h at t h e cl os ed fo rm o f M BP i s ab l e t o act i v at e s ub s t an t i al, b u t n o t m ax im al, ATP as e act i v i t y wi t h ou t ad op t i n g t h e op en c o n fo rm at i o n o r rel eas i n g i t s b o u n d s ub s t rat e. It h as b een d et erm i n ed t h at b o t h s t ab l e fo rm s o f M BP, m al t o s e-b o u n d -cl o s e d an d u n l i gan d ed -op en, s ep arat el y s t i m u l at e ATP h yd ro l ys is fro m t h e t r an s p o rt er. In co n t ras t, t h e s ub s t rat e i t s el f d o es n o t d i rect l y s t i m u l at e a ct i v it y b u t i n s t ead act i v at es h yd r o l ys i s b y s t ab i l i z i n g t h e cl o s ed s t at e o f M BP. Tak en t o get h e r o u r d at a in d i cat e t h at t h e t ran s p o rt er ex i s t s i n eq u i l i b ri u m b et ween an i n act i v e res t i n g s t at e an d co n fo rm at i o n s re cep t i v e t o b i n d i n g b y t h e cl o s ed an d op en fo rm s o f M BP. Th i s s u g ges t s t h a t un co up l ed ATP h yd ro l ys i s i s p rev en t ed b y d es t ab i l i z i n g ATP as e rel ev an t co n fo rm at i o n s o f t h e t ran s p o rt e r, an d t h at M BP act i v at es h yd ro l ys i s b y b i n d i n g t h e s e co n fo rm at i o n s i n i t s cl o s ed an d op en s t at es. Ke ywo rd s : Act i v e Tran s p o rt, ATP B i n d i n g C as s et t e, ATP as e, Escherichia Coli, C el l M em b ran e, M al t o s e Bi n d i n g P ro t ei n, M al t o s e Tran s p o rt, P ro t e i n C o n fo rm at i o n, S ub s t rat e S p eci fi ci t y, X-r a y C r ys t al l o grap h y iii

5 Co-authorship Chapter 2 M o d el i n g o f ex p ect ed s t i m u l u s k i n et i cs was p erfo rm ed b y Dr. Bri an S h i l t o n. S AXS ex p eri m en t s an d an al ys i s were p erfo rm ed b y Dr. Bri an S h i l t o n an d P at ri ck Tel m er. W o rk wi t h M BP m u t an t s M BP -DM an d M BP A9 6 W / I3 2 9 W was p er fo rm ed b y P at ri c k Tel m er. Th i s m an u s cri p t was co -wri t t e n b y D r. B ri an S h i l t o n, Pat ri ck Tel m er an d Al i s t er Go u l d an d ap p e ared i n Bi o ch em i s t r y, 2009 August 25; volume 48 (33) Th e m at e ri al i s rep ri n t ed h e re i n acco rd an c e wi t h AC S p o l i ci es. C op yri gh t A m eri can C h em i cal S o ci et y. Chapter 3: S AXS d at a wer e co l l ect e d an d an al yz ed b y Dr. Br i an S h i l t o n. Th i s m an u s cri p t was wri t t en b y Al i s t er Go u l d an d ed i t ed wi t h ad v i c e fr om Dr. Bri an S h i l t o n. It was p ub l i s h ed i n t h e J o u rn al o f Bi o l o gi c al C h em i s t r y April 9; volume 285(15) R ep ri n t ed wi t h p erm i s s i o n fro m AS BM B p ub l i cat i o n s. Chapter 4: P C R m u t agen es i s o f M B P -O9 5 / an d i n i t i al BM OE cros s l i n k i n g w as p erfo rm ed b y s u m m er s t u d en t Arj u n C h an d ra. S AXS d at a was co l l ect ed wi t h t h e as s i s t a n ce o f K ev i n Leu n g an d Dr. Bri an S h i l t o n. S AX an al ys i s was p erfo rm ed b y Dr. Bri an S h i l t o n. iv

6 Acknowledgments: I wo u l d l i k e t o t h an k Lee -An n B ri ere an d Dr. S t an l e y Ni t h i an an t h am fo r as s i s t an ce an d t rai n i n g i n p ro t ei n cr ys t al l i z at i o n, X-ra y d at a co ll e ct i o n an d s t ru ct u ral r efi n em en t. I t h an k Dr. M arc Os t e rm ei er an d Dr. Am y D av i d s o n fo r p ro v i d i n g v e ct o rs fo r s M BP a n d h i s t i d i n e t ag ged M al FGK 2. I t h an k P at ri ck Tel m e r fo r p as s i n g m e t h e t o rch o f a h i gh l y en g a gi n g res ear ch p roj ect, as wel l as Kev i n Leu n g an d Arj u n C h an d ra t h ei r co m p an io n s h i p an d as s i s t an ce. I wo u l d al s o l i k e t o t h an k m y ad v i s o r y co m m i t t ee, Dr. S t an Du n n an d Dr. J am es C h o y, t h ei r ad v i ce an d s up p o rt, as wel l as t h ei r t o l eran ce o f m y s o m et i m es op aq u e p r esen t at i o n s o f fi n d i n gs. I t h an k Dr. Bri an S h i l t o n, fo r o ff eri n g m e a p l a ce i n h i s l ab, m en t o ri n g m e t h ro u gh s o m e o f t h e fo rm at i v e ch al l en g es o f m y ed u c at i o n, an d giv i n g m e t h e t rem en d o u s b en efi t o f h i s ex p eri en ce. C r ys t al l o grap h i c d at a w e re co l l ect ed at t h e C an ad i an Li gh t S o u rc e, wh i ch i s s up p o rt ed b y Nat u ral S ci en c es an d En gi n e eri n g R es ea rch C o u n ci l of C an ad a, N at i o n al R es ear ch C o u n ci l, C an ad i an In s t i t u t es o f He al t h R es earch, an d t h e U n iv ers i t y o f S as k at ch ew a n. S AXS d at a wer e co l l ect e d at t h e Ad v an c ed P h o t o n S ou rce at Ar go n n e n at i o n al l ab s. Us e o f t h e Ad v an c ed P h o t o n S o u rce was s up p o rt ed b y t h e U.S. Dep art m en t o f En er g y, Of fi ce o f S ci en ce, Of fi ce o f B as i c En er g y S ci en c es, u n d er C o nt ract No. DE-AC C H an d W ENG-3 8. Th e Bi o C AT b eam l i n e i s s up p o rt e d b y N at i o n al In s t i t u t es o f Heal t h R es e arch Gran t R R I wo u l d al s o l i k e t o t h an k Fran ces an d Gra ce H a y, as wel l as Do u gl as, H eat h er, an d M eg an Go u l d fo r t h e i n c al cu l ab l e s up p o rt t h e y h a v e p ro v i d ed, wi t h o u t wh i ch n o n e o f t h i s wo rk wo u l d h av e b e en p o s s i b l e. v

7 Table of Contents Ab s t ract..i i i C o au t h o rs h i p..i v Ack n o wl ed gm en t s.....v Li s t o f Fi gu res....v i i i Li s t o f Tab l es.. i x Li s t o f Ab b r ev i at i o n s.. x Chapter 1 Introduction Back g ro u n d Th e M al t o s e Tran s p o rt er o f Escherichi Coli : En er g y Ut i l i z at i o n b y t h e ATP Bi n d i n g C as s et t e En er g y an d In fo rm at i o n Fl o w W i t h i n Th e Tran s m em b ran e S ub u n i t s R o l e o f t h e S ub s t rat e Bi n d i n g P ro t ei n Un an s wer ed Qu es t i o n s C o n cern i n g M al t o s e Tr an s p ort er Act i v i t y R es earch Go al s an d H yp o t h es i s Ex p eri m en t al M et h o d o l o g y S cop e o f Th es i s Bi b l i o gr ap h y Chapter 2 Stimulation of the Maltose Transporter ATPase by Unliganded Maltose Binding Protein In t ro d u ct i o n M at eri al s an d M et h o d s R es u l t s Di s cu s s i o n Bi b l i o gr ap h y Chapter 3 Studies of the Maltose Transport System Reveal a Mechanism for Coupling ATP Hydrolysis to Substrate Translocation Without Direct Recognition of Substrate In t ro d u ct i o n M at eri al s an d M et h o d s R es u l t s Di s cu s s i o n Bi b l i o gr ap h y Chapter 4 Activation of the Maltose Transporter by Cysteine Stabilized MBP Mutants In t ro d u ct i o n M at eri al s an d M et h o d s R es u l t s Di s cu s s i o n Bi b l i o gr ap h y vi

8 Chapter 5 General Discussion and Conclusions.169 Bi b l i o gr ap h y Appendix Appendix Appendix Curriculum Vitae vii

9 List of Figures Fi gu re 1.1 C o n s erv ed Do m ai n Ar ch i t ect u re o f A BC Tr an s p o rt ers 5 Fi gu re 1.2 C o n fo rm at i o n al C h an g es i n ABC S ub u n i t s on ATP Bi n d i n g... 9 Fi gu re 1.3 Ord er o f Ev en t s i n M al t o s e Tran s p o rt. 1 3 Fi gu re 1.4 P rop o s ed M ech an i s m o f C o up l i n g i n M al FGK Fi gu re 1.5 Th e S ub s t rat e Bi n d i n g S i t e an d B al an ci n g In t erf ac e Bal an ce C o n fo rm at i o n a l C h an ges i n M BP..2 5 Fi gu re 2.1 S t i m u l at i o n o f t h e M al FGK 2 ATP as e b y Un l i g an d ed M BP 4 8 Fi gu re 2.2 Ki n et i c An al ys i s o f M BP -M al FGK 2 ATP as e S t i m u l at i o n i n t h e P res en ce o f M al t o s e 5 0 Fi gu re 2.3 Ki n et i c An al ys i s o f M BP -M al FGK 2 ATP as e S t i m u l at i o n i n t h e Ab s en ce o f M al t o s e 5 3 Fi gu re 2.4 Ki n et i c M o d el s fo r S t i m u l at i o n o f M al FGK 2 ATP as e b y Op en an d C l o s ed M BP.5 8 Fi gu re 2.5 Ex p ect ed R at e C u rv es fo r M al FGK 2 S t i m u l at i o n b y M BP.6 1 Fi gu re 2.6 Un l i gan d ed M BP -A9 6 W / I3 2 9 W i s i n a C l o s ed C o n fo rm at i o n..6 5 Fi gu re 2.7 S t ab i l i t y o f C o n fo rm at i o n al l y En gi n e ered M BP M o l ecu l es Fi gu re 2.8 Des t ab i l i z at i o n o f t h e Op en C o n fo rm at i o n Dec re a s es M al FGK 2 ATP as e..7 0 Fi gu re 3.1 Op en t o C l o s ed C o n fo rm at i o n al C h an g e i n s M BP.8 6 Fi gu re 3.2 Effect o f Li g an d o n S t i m u l at i o n o f t h e Mal FGK 2 ATP as e. 8 9 Fi gu re 3.3 S u cro s e Bi n d i n g b y s M B P..9 3 Fi gu re 3.4 M ai n C h ai n Di s o rd er i n t h e Op en Un l i gan d ed s M BP S t ru ct u re.9 6 Fi gu re 3.5 Effect o f M u t at i o n s o n t h e S u rfac e P rop ert i es o f O p en an d C l o s ed s M BP.9 8 Fi gu re 3.6 M u t at i o n D1 4 L o f s M BP Fi gu re 3.7 In t e ra ct i o n s Bet w een t h e M al FGK 2 P 3 Lo op an d t h e s M BP Li g an d Bi n d i n g S i t e Fi gu re 3.8 S t ru ct u ral Bas i s fo r C o m m u n i cat i o n Bet we en t h e M al G P 3 Lo op an d ATP Bi n d i n g C as s et t es Fi gu re 4.1 M BP -C C ys t ei n e M u t at i o n s Fi gu re 4.2 ATP as e S t i m u l u s b y M B P -C i n P ro t eo l i p o s om es Fi gu re 4.3 ATP as e S t i m u l u s b y M B P -C o f Det er gen t S t ab i l i z ed M al FG K Fi gu re 4.4 M al t o s e Bi n d i n g M eas u r ed b y In t ri n s i c Fl u o r es ce n ce Qu en ch i n g Fi gu re 4.5 C h an ges i n S AXS P ro fi l e o f M BP -C wi t h M al t o s e Fi gu re 4.6 M BP -O C ys t ei n e M u t at i o n s Fi gu re 4.7 Fl u o res c en e La b el i n g o f BM OE C ro s s - Li n k ed M BP -O Fi gu re 4.8 M A LD I-TO F P ro fi l e o f C ro s s - Li n k ed M BP -O an d M BP -O9 5 / Fi gu re 4.9 ATP as e S t i m u l u s b y M B P -O i n P ro t eo l i p o s om es Fi gu re ATP as e S t i m u l u s b y M B P -O o f Det e r gen t S t ab i l i zed M al FG K Fi gu re C h an ges i n S AXS P ro fi l e o f M BP -O an d M BP - O 9 5 / wi t h M al t o s e viii

10 List of Tables Tab l e 2.1 Ki n et i c P aram et e rs fo r A ct i v at i o n o f M al FGK2 b y Li gan d ed an d Un l i gan d e d M BP.5 4 Tab l e 2.2 R at e C o n s t an t s fo r Ki n et i c M o d el s.5 9 Tab l e 3.1 C r ys t al l o grap h i c S t at i s t i cs..9 1 Tab l e 4.1 M u t agen i c P ri m ers fo r M BP M u t an t s Tab l e 4.2 R ad i i o f G yr at i o n o f M B P -C fro m GNOM An al ys i s Tab l e 4.3 M A LD I-TO F An al ys i s o f P rep ar ed Bi n d i n g P ro t e i n s Tab l e 4.4 R ad i i o f G yr at i o n o f M B P -O an d M BP -O9 5 / 1 71 f ro m GNOM An al ys i s ix

11 List of Abbreviations Å An gs t ro m ABC ATP Bi n d i n g C as s et t e ATP Ad en o s i n e Tri -P h o s p h at e BM OE 1,2 Bi s -m al ei m i d o -et h an e Da Dal t o n s DM S O Di m et h yl S u l fo x i d e DTT Di t h i o t h rei t o l EDTA Et h yl en e -d i am i n e-t et r a- a cet i c a ci d E. coli Escherichia Coli FM Fl u o res c ei n M al ei m i d e IP TG Is op rop yl β -D-1 -t h i o gal a ct op yr an o s i d e K D S ub s t rat e co n cen t r at i o n a t h al f m ax i m al b i n d i n g K m S ub s t rat e co n cen t r at i o n a t h al f m ax i m al act i v i t y M al E Gen e En co d i n g E. coli M BP M al FGK 2 M al t o s e Tran s p o rt e r o f E. coli M al FG Tran s m em b ran e s ub u n i t s o f E. coli m al t o s e t ran s p o rt er M al K ABC s ub u n i t s o f E. coli m al t o s e t ran s o rt er M A LD I-TO F M at ri x As s i s t ed Ti m e o f Fl i gh t m as s s p e ct ro m et r y M BP M al t o s e Bi n d i n g P ro t ei n wt M BP W i l d T yp e M BP M BP -DM M BP wi t h a b al an ci n g i n t erfa ce d el et i o n M BP -A9 6 W / I3 2 9 W M BP wi t h b u l k y r es i d u es i n s ert ed i n b al an ci n g i n t erfa ce M BP -C C l o s ed -S t ab i l i z ed M BP M u t an t M BP -O Op en -S t ab i l i z ed M BP M u t an t, BM OE M o d i fi ed M BP -O9 5 / Op en -S t ab i l i z ed M BP M u t an t, BM OE M o d i fi ed NEM N-Et h yl M al ei m i d e P BP P eri p h eral S ub s t rat e Bi n d i n g P ro t ei n P DB P ro t ei n Dat a B an k P gp P -gl ycop ro t ei n P LS P ro t eo l i p o s o m e S AXS S m al l An gl e X- ra y S cat t eri n g S BP S ub s t rat e Bi n d i n g P ro t ei n S DS -P AGE S o d i u m Do d ec yl S u l fat e P o l yacr yl am i d e Gel El e c t rop h o res i s s M BP S u cro s e Bi n d i n g M BP M u t an t TEV Tob acco Et ch Vi ru s TM Tran s m em b ran e Tri s Tri s -h yd ro x ym et h yl -am i n o m et h an e V ma x M ax i m al act i v i t y r at e wi t h ex ces s s ub s t rat e x

12 Chapter 1 Thesis Introduction 1.1 Background Li v i n g cel l s a re b o u n d ed b y s em i -p erm eab l e m em b ranes co m p o s ed o f l i p i d s, p ro t ei n s an d s u ga rs. Th ro u gh t h i s m e m b ran e t h e c el l i s ab l e t o res t ri ct, an d t o a d e gre e co n t ro l, i t s co m m u n i cat i o n s wi t h t h e o u t s i d e wo rl d. Bi o l o gi c al m em b ran es o f t h i s s o rt fu l fi l l t h ei r p u rp o s e b y r es t ri ct i n g t h e p as s a g e o f b u l k y o r s t ro n gl y h yd rop h i l i c m o l ecu l es ac ro s s t h ei r o i l y i n t eri o r l a ye r. Th i s cr eat es a p ro b l em fo r t h e c el l b ecau s e t h e n eed t o ret ai n an d p r o t ect d es i rab l e co m p o u n d s wi t h i n i t i s i n co n fl i ct wi t h t h e n eed t o t ak e i n n u t ri en t s an d ex p el was t e. To m e et t h i s n eed cel l s h av e d ev el op ed a v ari et y o f t ran s p o rt s ys t em s, m ac ro m o l ecu l ar s t ru ct u res em b ed d ed wi t h i n t h ei r c e l l m em b ran es, wh i ch al l o w th em t o m o d i f y t h ei r p erm eab i l i t y t o s p eci fi c s o l u t es. Th e m o s t co n cep t u al l y s i m p l e o f t h es e ar e p as s i v e t ran s p o rt ers ; p o res an d ch an n el s wh i ch p erfo rat e t h e b arri er o f t h e m em b r an e t o al l ow a fr eer ex ch an g e o f m at eri al s. Ex am p l es can ran g e i n co m p l ex i t y f ro m s i m p l e p o res l i k e s o m e aq u ap o ri n s [ 1,2 ], wh i ch al l o w t h e fl o w o f wat er as wel l as a d i v e rs i t y o f l a r ger m o l e cu l es, to ex q u i s i t el y s op h i s t i cat ed i o n ch an n el s ab l e t o d i s t i n gu i s h an d s el ect am o n g at o m s b as ed o n t h ei r si z e an d ch ar ge s t at e [ 3 ]. Th es e t ran s p o rt ers can b e s el ect i v e o f wh at m o l ecu l es t h e y p erm i t t o pas s an d can p erm i t o r p r ev en t p as s a g e i n res p o n s e t o v ar yi n g co n d i t i o n s, b u t al l p as s i v e t ran s p o rt ers ar e l i m i t ed b y e n t rop y. W i t h o u t t h e ex p en d i t u re o f en er g y i t i s n o t p o s s i b l e t o co n cen tr at e a co m p o u n d a gai n s t i t s t en d en c y t o d i ffu s e fro m are as o f h i gh co n ce n t rat i o n t o areas o f rel at i v e s car ci t y. Un l i k e p as s i v e s ys t em s, act i v e t r an s p o rt ers are a b l e t o accu m u l at e co m p o u n d s at rel at i v el y h i gh co n c en t rat i o n s b y co up l i n g t h e m o v em en t o f t h ei r t ar g et ed s u b s t rat e t o t h e ex p en d i t u re o f s o m e fo r m o f en er g y. Th es e t ran s p o rt ers h arn es s an ex i s t i n g en er g y r es erv e t o d ri v e a p ro ces s t h at wo u l d o t h erwi s e b e d i s f av o re d b y en t r op y. S t ru ct u ral l y, t h i s i s t yp i c al l y 1

13 acco m p l i s h ed b y a s m al l o ccl u d ed s p a ce wi t h i n t h e tran s p o rt e r, h arb o ri n g a s ub s t rat e b i n d i n g s i t e, t h at i s d ri v en b y co n fo rm at i o n al ch an g es t o al t ern at i v el y ac ces s ei t h e r f ace o f t h e m em b ran e [ 4,5,6 ]. An act i v e t ran s p o rt er c an gen e rat e a s ub st rat e gr ad i en t b y ex p o s i n g a h i gh - affi n i t y s i t e o n o n e m em b ran e fa ci n g fo r b i n d i n g an d a l o w- aff i n it y s i t e o n t h e op p o s i t e faci n g fo r d i s s o ci at i o n. S o m e act i v e t ran s p o rt e rs u t i l i z e an el ect ro ch em i c al grad i en t b as ed o n ex i s t i n g d i fferen ces i n ch ar ge d i s t ri b u t i o n an d s ub s t rat e co n cen t rat i o n. M an y t r an s p o rt ers l i k e t h e gl yc ero l -3 -p h o s p h at e t ra n s p o rt er Gl p T [ 7 ], arab i n o se i m p o rt er Ar ae [ 8 ], an d l act o s e p erm eas e La cy [ 9 ] h arn es s a t ra n s m em b ran e p ro t o n gr ad i e n t. Th e en er g y s t o r ed i n t h i s co m b i n ed p H an d ch ar ge g rad i en t i s r efe rre d t o as t h e p ro t o n m o t i v e fo rce an d i s u s ed b y t ran s p o rt ers o f t h e m aj o r faci l i t at o r s up er fam i l y. Ot h er act i v e t ran s p o rt e rs h arn es s t h e c el l s co m m o n en er g y cu rren c y, ad en o s i n e t ri -p h o s p h at e ( ATP ). ATP s t o res en e r g y i n p h o s p h o an h yd ri d e b o n d s b et ween i t s t h ree p h o s p h at e gro up s an d i n i t s ab u n d an ce r el at i v e t o it s b reak d o wn p ro d u ct s. Th e h yd ro l ys i s o f p h o s p h at e fro m ATP i s t h erefo re en er g et i cal l y fa v o rab l e, an d can b e u s ed t o d ri v e u n fav o r ab l e react i o n s. Th i s i s t h e en e rg y res e rv e u s ed b y t h e ATP Bi n d i n g C as s et t e ( ABC ) s up e rfam i l y o f act i v e t ran s p o rt ers. ABC t ran s p o rt ers ar e a fa m i l y o f act i v e t ran s p o rt e r s wi t h rep res en t at i v es i n ev er y s p e ci es [ 1 0,1 1 ]. Th es e co m p l ex es co up l e t h e t ran s m em b r an e t ran s p o rt o f a d i v ers e co l l ect i o n o f co m p o u n d s t o t h e h yd ro l ys i s o f ATP wi t h i n t h e ce l l. Th e fam i l y w as t erm e d i n res p o n s e t o t h e fi n d i n g t h at a s ub s et o f t h e h u m an m u l t i d ru g e ffl u x p u m p P -gl ycop ro t ei n ( P gp ), an d l at e r m an y o t h er eu k a r yo t i c ex p o rt er s, h el d ap p ro x i m at el y 3 0 % s eq u en c e h o m o l o g y t o a co l l ect i o n o f b i n d i n g p ro t ei n d ep en d en t b act eri al i m p o rt ers [ 1 2 ]. Th e fam i l y was fo u n d t o s p an fro m h u m an t ran s p o rt ers l i k e P gp, wh i ch i s res p o n s i b l e fo r d ru g r es i s t an ce i n m an y can cers, t o b a ct eri al m et ab o l i t e i m p o rt s l i k e th e m o l yb d at e t ran s p o rt e r Mo d BC an d m al t o s e t ra n s p o rt er M al F GK 2 2

14 [ 1 3,1 4,1 5 ]. Th e s eq u en ce h o m o l o g y o f t h es e t ran s p ort ers s t em s fro m t h e c o n s erv ed c yt op l as m i c ATP b i n d i n g cas s et t es t h at gi v e t h e p ro t ei n f am i l y i t s n am e. Th es e d o m ai n s can b e s u f fi ci en t l y s i m i l ar i n s t ru ct u re an d f u n ct i o n t h at t h e y h av e b e en ex ch an ged b et w een d i fferi n g t r an s p o rt ers wi t h o u t ab ro g at i n g t h ei r fu n ct i o n [ 1 6 ]. Th e s eq u en ce h o m o l o g y b et ween A BC t ran s p o rt er s i s l i m i t ed t o t h e ABC sub u n i t s, b u t t h e fam i l y n o n et h el es s m ai n t ai n s a h i gh l y co n s erv ed d o m ai n s t ru ct u re (Fi g. 1.1 ). Al t h o u gh t h e v ari o u s d o m ai n s o f t h e c o m p l ex are s o m et i m es fu s ed t o get h er i n a s m al l e r n u m b er o f p ep t i d e s t ran d s, t h e n u m b er an d gen eral fu n ct i o n o f e ach d om ai n i s m ai n t ai n ed. Each A BC t ran s p o rt e r i n co rp o rat es a h et e ro - o r h o m o - d i m er o f t ran s m e m b ran e (TM ) d o m ai n s, e m b ed d ed wi t h i n an d s p an n i n g t h e m em b ran e. Th e TM d o m ai n s are p ri m ari l yα-h el i cal an d s erv e t o an ch o r t h e co m p l ex, fo rm t h e t ran s l o cat i o n p o re i t s el f an d ac co u n t fo r m u ch o r al l o f t h e co m p l ex s s ub s t rat e s p eci fi c fu n ct i o n. In ad d i t i o n t o t h e TM re gi o n s an d an ch o red t o t h em, b y t i gh t b i n d i n g an d / o r d i rect p ep t i d e b o n d s, are t h e n am es ak e AB C sub u n i t s. Each co m p l ex i n co rp o rat es a h o m o - o r h et ero -d i m er o f ABC s ub u n i t s o n t h e c yt op l as m i c faci n g o f t h e m em b ran e, t h u s al l o wi n g a cc es s t o t h e cel l u l ar p o o l o f ATP. P ro k ar yo t i c i m p o rt ers ad d i t i o n al l y i n co rp o r at e a p eri p h er al s ub s t rat e b i n d i n g p ro t ei n (P BP ). Th e P BP i s fo u n d o n t h e ex t eri o r fac e o f t he m em b ran e, o ft en fl o at i n g fr ee i n t h e p eri p l as m o f Gr am n e gat i v e b act eri a o r as s o ci at ed wi t h t h e m em b ran e ex t er i o r. Th e P BP act s as t h e i n i t i al b i n d i n g s i t e fo r s ub s t rat e b efo re i n t er act i n g wi t h t h e TM d o m ai n s o f t h e t ran s p o rt e r. Th i s i n t eract i o n b et we en b i n d i n g p ro t ei n an d s ub s t rat e i s n ec es s ar y fo r t h e a ct i v at i o n o f ATP h yd ro l ys i s an d s ub s t rat e t ran s p o rt, as A BC i m p o rt ers d o n o t ap p e ar t o act o n s ub s t rat e t h at h as n o t fi rs t b een b o u n d t o a P BP [ 2 0,2 1 ]. 3

15 Fig 1.1 Conserved Domain Architecture of ABC Transporters Th e X-r a y cr ys t al s t ru ct u res o f s ev eral A BC t ran s po rt ers ar e s h o wn. D es p i t e act i n g o n d i fferen t s ub s t r at es, t h e s t ru ct u res r ev eal co m m o n arch i t ect u ral fe at u res i n t h e ABC s ub u n i t s (g reen an d c ya n ), t h e t r a n s m em b ran e r e gi o n s (p i n k, yel l o w ) an d t h e s ub s t r at e b i n d i n g p ro t ei n (m ag en t a). No t e t h at S A V i s a s ub s t rat e ex p o rt er an d d o es n o t h av e a n as s o ci at ed s ub s t rat e b i n d i n g p ro t ei n. P DB ref eren c e: 2 R 6 G[ 1 7 ], 2 ONK[ 1 5 ], 2 QI9 [ 1 8 ], 2 HYD[ 1 9 ] 4

16 5

17 Du e t o t h i s d o m ai n arran gem en t, b i n d i n g p ro t ei n d epen d en t A BC i m p o rt er s i n co rp o rat e a s eri es o f f eat u r es t h at m a k e t h em v al u ab l e s ub j e ct s o f s t u d y t o b ro ad en o u r u n d ers t an d i n g o f p ro t ei n co m p l ex en gi n e er i n g. A s i gn al t ran s d u ct i o n m ech an i s m i s req u i red t o co m m u n i cat e t h e p res en c e o f s ub s t rat e o u t s i d e t h e cel l t o t h e cel l i n t eri o r, s o t h at ATP h yd ro l ys i s can b e i n i t i at ed. Ad d i t i o n al l y, an en er g y t ran s d u ct i o n m ech an i s m i s neces s a r y t o co n v e rt A TP h yd ro l ys i s wi t h i n t h e c yt op l as m i n t o t h e d i rect ed t ran s p o rt o f t h e r el a t i v el y d i s t an t t ar get s ub s t rat e. Th e TM d o m ai n s are r eq u i red b o t h t o fo rm t h e t ran s l o cat i o n p at h wa y an d al s o t ran s m i t t h es e co m m u n i cat i o n s b et ween t h e ABC s an d P BP. 1.2 The Maltose Transporter of Escherichia coli: Th e m ech an i s m s b y wh i c h ABC t ran s p o rt ers t ran s d u ce i n fo rm at i o n an d en e rg y b et ween t h ei r s ub u n i t s are i n t ri gu i n g s ub j ect s o f res ear ch, b o t h as s t u d i es i n en z ym e en gi n eeri n g an d b ecau s e o r gan i s m s rel y o n t h es e s ys t em s fo r t h e u p t ak e an d s e cret i o n o f v i t al n u t ri en t s an d t o x i n s. Thi s t h es i s s eek s t o b ro ad en o u r u n d ers t an d i n g o f t h es e m ech an i s m s b y s t u d yi n g t h e ATP as e re gu l at i o n o f t h e m al t o s e t ran s p o rt er o f Escherichia coli ; M al FGK 2. Th i s i s o n e o f t h e m o s t s t u d i ed ABC t ran s p o rt ers an d i s co n s i d e red a m o d el s ys t em fo r b i n d i n g-p ro t ei n -d ep en d en t ABC t ran s p o rt d u e t o t h e co n s erv ed d o m ai n arch i t ectu re o f t h e fam i l y [ 2 2 ]. S ev eral f eat u res m ak e i t an ap p e al i n g s ub j ect o f s t u d y. Th es e i n cl ud e t h e t ran s p o rt e r s n e ces s ar y ro l e i n m al t o s e m et ab o l i s m, wh i ch en ab l es res e arch ers t o as s a y t r ans p o rt b y ob s erv i n g t h e gro wt h co m p et en c y o f cel l s o n m al t o s e b as ed m ed i a, as wel l as t h e ad op t i o n o f i t s P BP as a res earch t o o l i n p ro t ei n s ecret i o n an d en gi n ee ri n g s t u d i es [ 2 3 ]. Du e t o t h es e s t u d i es t h ere i s a wea l t h o f i n fo rm at i o n o n P BP s t ru ct u re, co n fo rm at i o n al d yn am i cs an d s ub s t rat e b i n d i n g act i v i t y wh i ch can b e u s ed t o i n fo rm ex p eri m en t s o n M al FGK 2. 6

18 Th e m al t o s e t ran s p o rt e r s h ares t h e co m m o n d o m ai n arch i t ect u re o f o t h e r A BC t ran s p o rt ers a n d ea ch o f i t s d o m ai n s co n s i s t s o f a s ep arat e p o l yp ep t i d e s t ran d (Fi g. 1.1 ). It s TM d o m ai n s are co m p o s ed o f a h et e ro d i m er fo rm ed b y t h e M al F an d M al G s u b u n i t s, wh i l e i t s ABC s are a h o m o d i m e r o f 2 M a l K s ub u n i t s. It s co gn at e P BP i s t h e m al t o s e b i n d in g p ro t ei n (M BP ), wh i ch fl o at s fr eel y i n t h e p eri p l as m (Fi g 1.2 ). At t h e i n i t i at i o n o f t h i s t hes i s a g en er al s eq u en ce o f ev en t s i n m al t o s e t ran s p o rt was u n d ers t o o d. It was k n o w n t h at m al t o s e en t ers t h e peri p l as m o f t h e c el l t h ro u gh t h e Lam B m al t o s e p o re b y fa ci l i t at e d d i ffu s i o n [ 2 4 ]. On t h e p eri p l as m i c s i d e o f t h e o u t er cel l m em b ran e m al t o s e i s b o u n d an d s eq u es t ered b y M BP. Th e b i n d i n g o f m al t o s e s t ab i l i z es a s ub s t an t i al co n fo rm at i o n al ch an ge i n M BP fro m a r es t i n g op en s t at e t o an al t ern at i v e cl o s ed s t at e (Fi g 1.2 ) [ 2 5,2 6,2 7 ]. In t h e p r es en ce o f m al t o s e, M BP i s ab l e t o s t i m u l at e rap i d ATP h yd ro l ys i s an d m al t o s e i n t ern al i z at i o n b y t h e t r an s p o rt er (Fi g 1.2 ). S ub s eq u en t t o t h e b egi n n i n g o f t h i s t h es i s, t h ree X -ra y c r ys t al s t ru ct u res o f t h e m al t o s e t ran s p o rt er co m p l ex h av e b een res o l v ed ad op t i n g d i fferen t co n fo rm at i o n s t h o u gh t t o rep res en t s t ages o f t h e t ran s p o rt c ycl e [ 2 8,1 3,1 7 ]. Th es e s t ru ct u res i n d i cat e t h at t ran s p o rt b y t h e m al t o s e t ran s p o rt er i n v o l v es a s er i es o f ri gi d b o d y ro t at i on s t h at s erv e t o co n v e rt t h e TM d o m ai n s fro m a c yt op l as m i c faci n g r es t i n g s t at e t o a p eri s p l as m i c f aci n g s t at e an d b ack ( Fi g 1.2 ). Th i s c ycl e h as b een t erm ed t ran s p o rt b y al t ern at i n g a cces s [ 2 8 ]. C o n v ers i o n fro m t h e res t i n g s t at e t o t h e p eri p l as m i c fa ci n g s t at e i s l i n k ed t o cl o s u re o f t he ABC s ub u n i t s an d an op e n i n g o f t h e as s o ci at ed M BP, s u ch t h at m al t o s e can p as s i n t o t h e TM re gi o n s t h ro u gh a n o ccl u d ed t ran s p o rt p as s a ge [ 1 7 ]. Th i s co n fo rm at i o n i s ad d i t i o n al l y t h o u gh t t o b e t h e ATP as e c at al yt i c s t at e o f t h e co m p l ex b ecau s e i t was o b s erv ed b y a rres t i n g AT P h yd ro l ys i s t h ro u gh an ATP b i n d i n g s i t e p o i n t m u t at i o n. ATP h yd ro l ys i s i s b el i ev ed t o fo rc e t h e s ep arat i o n o f t h e ABC s u b u n i t s an d ret u rn t h e 7

19 Fig 1.2 Order of Events in Maltose Transport M al t o s e en t ers t h e p eri p l as m t h ro u gh t h e La m B m al top o ri n. In t h e p eri p l as m, m al t o s e i s b o u n d b y M BP, wh i ch s h i ft s fro m an op en t o cl o s e d co n fo rm at i o n. In t h e cl o s ed fo rm, M BP i s ab l e t o act i v at e M al FGK 2, res u l t i n g i n t h e co n v ers i o n o f t h e t ran s p o rt er fro m t h e res t i n g s t at e t o t h e ATP as e c at al yt i c s t at e. Aft er t h e p as s a g e o f m al t o s e i n t o t h e TM s ub u n i t s, t h e h yd ro l ys i s o f ATP d es t ab i l i z es t h e cat a l yt i c s t at e an d res u l t s i n a rev ers i o n t o t h e r es t i n g s t at e, wi t h res u l t i n g d i s s o ci at i o n o f m al t o s e i n t o t h e c yt op l as m. 8

20 9

21 co m p l ex t o i t s res t i n g s t at e [ 2 9 ]. 1.3 Energy Utilization by the ATP Binding Cassette: Th e ATP b i n d i n g c as s et t es o f A BC t ran s p o rt ers ar e t h e p ri m ar y s o u r ce o f e n er g y wi t h i n t h es e co m p l ex es ; wh ere ATP i s b o u n d an d h yd ro l yz ed, c au s i n g ch an ges i n en er g y t h at d ri v e u s efu l co n fo rm at i o n al ch an g es wi t h i n t h e l ar ge r c o mp l ex [ 3 0,3 1 ]. W h at i s k n o wn ab o u t co n fo rm at i o n al ch an ges i n t h e ABC s o f t h e m al t o s e tran s p o rt e r cam e i n i t i al l y fro m h i gh - res o l u t i o n X-ra y s t ru ct u r es o f t h e i s o l at ed M al K s ub u n i t s i n s o l ub l e d i m ers [ 3 2,3 3 ]. Ho wev er, t h es e fi n d i n gs h av e b e en i n creas i n gl y v al i d at ed i n X-ra y s t ru ct u res o f fu l l ABC t ran s p o rt e r co m p l ex es [ 1 7,2 8,1 3]. Th e M al K s ub u n i t s o f t h e m al t o s e t ran s p o rt e r d i s p l a y a s t ru ct u re wi t h b ro a d s i m i l ari t i es t o o t h er ABC s an d ad d i t i o n al l y i n co rp o rat e a co l l e ct i o n o f ri gi d l y co n s erv e d ABC s eq u en ce feat u r es. Am o n g t h es e f eat u res are t h e W al k e r A and B ATP -b i n d i n g m o t i fs co m m o n t o a n u m b er o f ATP b i n d i n g en z ym es [ 3 4 ]. Each M al K al s o co n t ai n s an ab s o l u t el y co n s erv ed s i gn at u r e s eq u en ce i n co rp o rat i n g t h e s eq u en ce LS GGQ, wh i ch i s u n i q u e t o t h e ABC t ran s p o rt er f am i l y an d u s ed t o d efi n e i t s m em b ers [ 3 5 ]. Du ri n g act i v i t y, t h e W al k er A an d B m o t i fs o f e ac h M al K co -o rd i n at e b o t h t h e t erm i n al β an d γ p h o s p h at es o f an A TP m o l ecu l e an d a m a gn es i u m i o n. Th i s arran ge m en t al l o ws fo r t h e s ep arat e b i n d i n g o f a m o l ecu l e o f A TP t o each A B C sub u n i t. W h en t h e Mal K d i m er i s i s o l at ed i n s o l u t i o n o r i n co rp o rat e d i n t o an M BP s t i m u l at ed t ran s p o rt er co m p l ex, t he b i n d i n g o f ATP cau s es i t t o re-o r gan i z e, wi t h t h e t wo s ub u n i t s m ore cl o s el y as s o ci at ed. A ro t at i o n o f t h e t wo s ub u n i t s, wi t h res p ect t o each o t h e r, al l o ws t h e LS GGQ m o t i f o f ea ch M al K t o co n t act t h eγ p h o s p h at e o f t h e ATP b o u n d t o t h e op p o s i n g s ub u n i t, fo rm i n g a co m p o s i t e ATP -b i n d i n g s i t e 10

22 t h at i n co rp o rat es res i d u es fro m ea ch s ub u n i t (Fi gu r e 1.3 ). Th i s ro t at i o n h as b een t erm ed a t weez ers l i k e m o t i o n af t er t h e wa y i n wh i ch t h e two s ub u n i t s are d ra wn cl o s er t o g et h er [ 3 2 ]. ATP h yd ro l ys i s o ccu rs i n t h i s s t at e, wi t h each AT P co -o rd i n at ed b y b o t h M al K s ub u n i t s [ 1 7]. No t e t h at o n e o f t h e m o r e en er g et i c co n t a ct s i n t h is d i m er co n fo rm at i o n i s a 1.9 Å h yd ro gen b o n d b et ween S eri n e o f t h e s i gn at u re s eq u en ce an d an o x yg en at o m o f t h e op p o s i n g ATP γ p h o s p h at e [ 3 6,3 2 ]. Fo l l owi n g cl eav a g e o f ATP t o ADP d u ri n g h yd ro l ys i s, an d rel e as e o f t h eγ p h o s p h at e, t h i s b o n d i s n o l o n ger p o s s i b l e an d l i k el y acco u n t s fo r a s i gn i fi can t p o rt i o n o f t h e en er get i c d i ffe ren c es b et ween A BC co n fo rm at i o n s, ex p l ai n i n g wh y ATP a n d n o t ADP s up p o rt s ABC d i m er cl o s u r e [ 3 7,3 8 ]. Th i s co m p o s i t e b i n d i n g-s i t e cau s es s ub s t an t i al, an d en er get i c, co n fo rm at i o n al ch an ges i n t h e ABC d o m ai n s t o b e m ad e co n t i n gen t o n t h e b i n d i ng an d h yd ro l ys i s o f ATP (Fi g 1.3 ). Th es e co n fo rm at i o n al ch an ges t ak e p l ac e i n t h e co n t ex t o f a t i gh t as s o ci at i o n b et w een t h e A BC d i m er an d t h e TM s ub u n i t s, n ec es s i t at i n g an i n fl u en ce o f each o n t h e o t h er. ATP b i n d i n g an d h yd ro l ys i s m u s t co m m u n i cat e en er g y, i n t h e fo rm o f co n fo rm at i o n al ch an g es, i n t o t h e at t ach ed TM d o m ai n s. Ad d i t i o n al l y, t h e r el at i v e ri gi d i t y o r fl ex i b i l it y o f t h e TM r e g i o n s l i k el y p l a ys a ro l e i n co n t ro l l i n g t h e A BC s ab i l i t y t o cl o s e aro u nd an d h yd ro l yz e ATP. 1.4 Energy and Information Flow Within The Transmembrane Subunits Th e t ran s m em b r an e (TM ) s ub u n i t s o f ABC t ran s p o rt ers co n s t i t u t e t h e b u l k o f t h e act u al t ran s p o rt m ach i n e r y o f t h e co m p l ex. In A BC ex p o rt ers, wh i ch c o n t ai n n o o t h er s ub s t rat e- b i n d i n g s i t e, t h e TM s ar e res p o n s i b l e fo r s ub s t rat e reco gn i t i o n an d s el e ct i v i t y. In b i n d i n g p ro t ei n -d ep en d en t i m p o rt ers, l i k e t h e m al t o s e t ran s p o rt er, t h e TM s ub u n i t s m u s t s t i l l reco gn i z e t h e co gn at e P BP an d res p o n d t o t h e m o v em en t o f s ub st rat e i n t o t h e t ran s l o c at i o n p at h wa y. Th e 11

23 Fig 1.3 Conformational Changes in ABC Subunits on ATP Binding Th ree X -ra y cr ys t al s t ru c t u res are s h o wn o f t h e m al t o s e t ran s p o rt er o f E. coli wi t h t h e t ran s m em b ran e d o m ai n s (p i n k, yel l o w ) i n -co m p l ex wi t h i t s s ub s t rat e b i n d i ng p ro t ei n (m a gen t a ) an d ABC s ub u n i t s ( gre en, c ya n ). Bl ack l i n es d en o t e t h e cel l m em b ran e an d t h e s ub s t rat e m al t o s e i s s h o wn wi t h i n t h e co m p l ex. As t h e cat al yt i c c ycle o f t h e t r an s p o rt er p ro gr es s es, an d wi t h t h e b i n d i n g o f ATP, t h e A B C s ub u n i t s u n d ergo co n f o rm at i o n al ch an ges fro m an op en s t at e t o a s em i -op en an d u l t i m at el y cl o s ed s t at e (r ed wed g e). Th i s cl o s u re en ab l es t h e fo rm at i o n o f a co m p o s i t e b i n d i n g s i t e fo r each ATP m o l ecu l e ( re d rect an gl es ) an d i s co m m u n i cat ed t h ro u gh t h e co up l i n g l o op s (ci r cl es ) t o d ri v e co n fo rm at i o n al c han g es i n t h e res t o f t h e c o m p l ex. P DB refe ren c e: 3 FH6 [ 2 8 ], 3 P VO[ 1 3 ],2 R 6 G [ 1 7 ] 12

24 13

25 s eq u en ces o f TM re gi o n s wi t h i n t h e ABC s up erf a m i l y h av e d i v e r ged s o b r o ad l y b ecau s e o f t h e n eed t o act o n s ub s t rat es o f d i v ers e s t ru ct u re. Not e t h at d es p i t e t h i s d i v ers i t y o f s eq u en c e, p h yl o gen et i c an al ys i s i n d i cat es a co n s i s t en t p at t e r n o f d es cen t am o n g P BP s, TM s an d ABC s [ 3 9,4 0 ]. Th is i n d i cat es t h at t h e t ran s p o rt e rs h av e b een ad ap t ed wh o l e f ro m an an c es t ral co m p l ex an d are t h u s m u ch m o r e l i k el y t o s h ar e co m m o n m ech an i s m s t h an i f t h e y h a d b een b u i l t fro m n o v el co m p o n en t s. C o n s i s t en t wi t h t h i s fi n din g, t h e TM s ub u n i t s m ai n t ai n b ro ad l y s i m i l arα- h el i cal s t ru ct u res an d a h i gh l y c o n s erv ed i n t er fac e wi t h t he ABC s ub u n i t s, d es p i t e t h ei r v ari ed s eq u en c e. Each TM s ub u n i t i s t i gh t l y b o u n d t o i t s as s o ci at ed ABC b y m ean s o f a co n s erv ed f eat u r e k n o wn as a co up l i n g l o op [ 1 5 ]. Th i s s h o rt α h el i x ru n s p aral l el t o t h e s u rf ace o f t h e m em b ran e, ex t en d s i n t o t h e c yt op l as m an d res t s wi t h i n a s t ru ct u ral l y co n s erv ed gro o v e o f t h e A BC. Th e co up l i n g l o op s m i m i c t h e ap p ear an c e an d fu n ct i o n o f a p ai r o f h an d l es o n t o wh i ch t h e A BC s are at t ach ed. Th e ar ran gem e n t co n v e ys t h e i m p res s i o n th at t h e ABC s ub u n i t s act as a p o w er s o u r ce op t i m i z ed t o d ri v e m an y s ys t em s i n a s i m i l ar fas h i on. Th e y ar e af fi x ed t o a d ev i ce t h at i s s h ap ed b y t h e n e ed s o f i t s s p eci fi c fu n ct i o n b u t m ai n t ai n s feat u r es n ec es s ar y t o ef fi ci en t l y d ra w p o wer. Th at t h e ABC s ub u n i t s v ar y s o l i t t l e i n s t ru ct u re m ean s t h at t h e TM re gi o n s m u s t ex erci s e co n s i d erab l e co n t ro l o v er t h ei r act i v i t y i n o rd er fo r each co m p l ex t o co up l e ATP h yd ro l ys i s t o t h e t ran s p o rt o f d i ff eren t l y co m p o s ed s ub s t rat es. In d eed, i n i s o l at i o n t h e M al K s ub u n i t s o f t h e m al t o s e t ran s p o rt er are a b l e t o d i m eri z e an d cl o s e o n t h e b i n d i n g o f ATP a s p rev i o u s l y d i s cu s s ed, res u l t i n g i n a s m al l d e g re e o f co n s t i t u t i v e ATP as e act i v i t y. Ho w ev er wh en M al K i s b o u n d t o t h e M al F an d M al G TM d o m ai n s, v i a t h e co up l i n g l o o p s, t h i s act i v i t y i s ab ro ga t ed an d ATP h yd ro l ys i s i s ren d ered co n t i n gen t o n a ct i v at i o n b y M BP [ 4 1,4 2 ]. Th e TM do m ai n s i n h i b i t t h e 14

26 ABC s ub u n i t s b y i m p o s i n g co n fo rm at i o n al o r en e rg et i c res t r ai n t s t h at m u s t b e rel i ev ed t o en ab l e act i v i t y [ 2 9 ]. Th i s au t o -i n h i b i t i o n i s p art i cu l arl y s t ri k i n g gi v en t h at ATP co n cen t rat i o n s i n t h e cel l are far i n ex ces s o f t h e K m o f m o s t ABC s ub u n i t s, l i k el y cau s i n g t h e A BC s t o beco m e s at u rat ed wi t h ATP ev en wh i l e i n a rel ax ed op en s t at e. W h en t ran s p o rt co up l ed ATP as e i s act i v at ed b y m al t os e b o u n d M BP, t h e T M d o m ai n s h av e t h e r ev ers e ef fect an d i n s t ead s t ab i l i z e o r act i v at e t h e A BC s t o a h i gh e r rat e o f h yd ro l ys i s t h an s een i n i s o l at i o n. A m aj o r m o t i v at o r o f t h e w o rk i n t h i s t h es i s i s t o i n ves t i gat e h o w t h e TM s egm en t s o f t h e t ran s p o rt er i n h i b i t t h e ABC s ub u n i t s, wh i ch are c ap ab l e o f co n s t i t u t i v e act i v i t y, an d t h en rev ers e t h at i n h i b i t i o n s o as t o s up p o rt an ev en h i gh er l ev el o f a ct i v i t y wh en s ub s t r at e i s av ai l ab l e. In M al FGK 2 t h e co m m u n i cat i o n b et ween s ub s t r at e an d A BC s ub u n it s i s co m p l i cat ed b y p eri p l as m i c M BP. Th e s ys t em r eq u i res s ub s t rat e t o b e p res en t ed b y M BP b efo re t r an s p o rt an d ATP h yd ro l ys i s ar e t ri g g ered. Th i s r eq u i res t h at t h e TM d o m ai n s m ed i at e co m m u n i cat i o n s b et ween M BP o n t h e ex t ern al f aci n g o f t h e m em b ran e an d ABC s o n t h e i n t ern al f ace [ 2 9,4 3 ]. Ho wev e r, a n u m b er o f m u t an t co m p l ex es h av e b e en i s ol at ed t h at i n co rp o r a t e p o i n t m u t at i o n s wi t h i n t h e TM regi o n s. Th es e t ran s p o rt ers a re ga i n-o f-fu n ct i o n m u t an t s t h at h av e b e en fr eed fro m d ep en d en ce o n M B P an d are ab l e t o ca rr y o u t t ran s p o rt i n i t s ab s en ce. Kn o wn as M BP - i n d ep en d en t m u t an t s, t h es e t ran s p o rt ers co n fi rm t he ro l e o f t h e TM re gi o n s i n m ed i at i n g i n fo rm at i o n fl o w b y act i v at i n g t h e ATP as e o f a w i l d t yp e A BC d i m er ex cl u s i v el y t h ro u gh m u t at i o n o f t h e TM d o m ai n s. An ad d i t i o n al i n fo rm at i v e feat u re o f t h es e m u t an t s i s t h at t h e y ar e ab l e t o o v erco m e M BP d ep en d en ce o n l y b y ad op t i n g an u n co up l ed p h en o t yp e. M u t an t s o f t h i s t yp e are ab l e t o t ran s p o rt m al t o s e, b u t d o s o at a ra t e t h at i s i n d i rect co rr el a t i o n t o t h e rat e o f u n co up l ed ATP h yd ro l ys i s 15

27 [ 4 4 ]. Th es e co m p l ex es are v er y l i k el y c ycl i n g t h r ou gh t ran s p o rt rel ev an t c o n fo rm at i o n s i n an u n co n t ro l l ed m an n er, u n co up l i n g ATP h yd ro l ys i s fro m m al t o s e t ran s p o rt. ATP i s h yd ro l yz ed co n s t i t u t i v el y, an d m al t o s e i s t ran s p o rt ed b y ch an ce. It t h er efo r e ap p ears t h at M BP p l a ys an i n t egr al ro l e i n s ub s t rat e reco gn i t i o n, as M BP d ep en d en c e can b e o v er co m e o n l y b y a l o s s o f ATP as e re gu l at i o n. 1.5 Role of the Substrate Binding Protein As p rev i o u s l y s t at ed, A B C i m p o rt ers l i k e t h e m al t o se t ran s p o rt e r req u i r e s u b s t rat e t o b e p res en t ed b y a p eri p l as m i c s ub s t rat e b i n d i n g p ro t e in. Th i s d ep en d en c y m a y b e l i n k ed t o p h yl o gen y, as i n d i cat ed b y t h e ex cl u s i v e ap p ear an ce o f A BC i m p o rt ers i n p ro k ar yo t es, wh i l e ex p o rt ers are co m m o n t o al l d o m ai n s o f l i fe. Li k e t h e TM d o m ai n s, t h e p eri p h eral s ub s t rat e b i n d i n g p ro t ei n s (P BP s ) o f ABC t ran s p o rt ers ex h i b i t l i t t l e o r n o s eq u en ce h o m o l o g y, yet m o s t s h are a b ro ad l y co n s erv e d s t ru ct u re. M an y b i n d i n g p ro t ei n s s h are a co m m o n fo l d i n g m o t i f, b as ed aro u n d t wo d o m ai n s co n n ect ed b y a fl ex i b l e h in g e re gi o n. Th e s ub s t rat e b i n d i n g s i t e i s l o cat ed wi t h i n a cl e ft at t h e d o m ai n i n t erfa ce [ 4 5,4 6,4 7,4 8 ]. Th i s i s an i n di cat i o n t h at t h e wa y i n wh i ch t h es e p ro t ei n s i n t e ract wi t h t h ei r co gn at e t r an s p o rt ers m a y al s o b e b r o ad l y s i m i l ar. Al t h o u gh i t i s wel l reco g n i z ed t h at t h e m al t o s e b in d i n g p ro t ei n i s n e ces s ar y fo r t h e act i v at i o n o f t h e m al t o s e t ran s p o rt er, i t s ad ap t i v e r o l e i n t ran s p o rt i s a m at t e r o f s o m e s p ecu l at i o n. It h as b e en s u g ges t ed t h at M BP al l o ws t h e s ub s t rat e t o b e d e - s o l v at ed p ri o r t o i n t eract i o n wi t h t h e t ran s p o rt er an d h en ce g reat l y acc el erat es t h e act o f t ran s p o rt as co m p ar ed t o a t ran s p o rt e r t h at m u s t o v erco m e h yd rat i o n en e r gi e s d u ri n g t r an s p o rt [ 4 5 ]. A h i gh - affi n i t y s ub s t rat e b i n d i n g s i t e o n t h e P BP m a y fre e t h e T M re gi o n s t o d ev el op a l o wer a ffi n i t y s i t e wh i ch wi l l m o re read i l y ex p el s ub s t rat e i n t o t h e c yt op l as m an d al l o w t h e m ai n t en an ce o f a l ar g er 16

28 s ub s t rat e grad i en t t h an i n t h e ab s en c e o f a P BP [ 4 9]. In d eed, wh en ex p o s e d, t h e s ub s t rat e b i n d i n g s i t e wi t h i n t h e TM re gi o n s o f A BC t ran s p o rt ers d o es h av e a l o w er affi n i t y fo r s ub s t rat e co m p ared t o t h at o f t h ei r co gn at e P BP s [ 5 0 ]. M BP i n d ep en d en t m al t o s e t r an s p o rt er m u t an t s ap p ear t o h av e a ffi n i t i es i n t h e l o w m M ran ge, as c o m p ared t o t h e 4 µ M affi n i t y o f M BP fo r m al t o s e [ 5 1 ]. Ad d it i o n all y, M BP a ct s as a s ub s t r a t e s en s o r fo r b o t h t h e m al t o s e t ran s p o rt er an d an o t h er s i gn al i n g co m p l e x, s u gges t i n g t h at P BP s m a y s o m et i m es e co n o m i z e gen e ex p res s i o n [ 5 2 ]. Fi n al l y, h i gh ex p re s s i o n o f M BP, wh i ch i s p res en t at 1 m M wi t h i n t h e p eri p l as m o f Escherichia coli can g en erat e an ex ces s o f s ub s t ra t e b i n d i n g s i t es, en ab l i n g t h e cel l t o rap i d l y cap t u re an d ret ai n m al t o s e fro m a n u t ri en t p o o r en vi ro n m en t [ 2 2 ]. Al l o f t h es e fa ct o rs ar e l i k el y at p l a y i n P BP s elec t i o n, b u t s ev eral o f t h es e ex p l an at i o n s h i n ge o n a co m m o n fe at u re; t h at m o v i n g t h e i n i t i al s ub s t rat e-b i n d i n g s i t e o u t o f t h e t ran s p o rt er an d i n t o a s o l ub l e p ro t ei n en ab l es i t t o b e ex p res se d i n far gre at er cop y n u m b er. P ack i n g co n s t rai n s l i m i t t h e ab i l i ty o f a cel l t o ex p res s pro t ei n s wi t h i n t h ei r m em b ra n e, as ex ces s i v e cro wd i n g i m p act s t h e fu n ct i o n o f b o t h p ro t ei n s an d m em b ran e [ 5 3,2 2 ]. Exp res s i o n o f P BP s i s o v er an o rd er o f m a gn i t u d e h i gh er t h an o f t h ei r as so ci at ed b i n d i n g p art n ers i n t h e m em b ran e, up t o 4.6 x M BP m o l ecu l es are t yp i c al l y p res en t kj fi n t h e p erip l as m o f E. coli, wh i l e o n l y ab o u t m al t o s e t ran s p o rt er co m p l ex es are em b ed d ed i n t h e i n n er m em b ran e [ 5 2,5 4,5 5,5 6 ]. Th i s eco n o m i z es t h e u s e o f m em b ran e s p ace wi t h o u t l i m i t in g t h e ab i l i t y t o b i n d s ub s t rat e, s u g g es t i n g t h at t h i s m a y b e a d ri v i n g co n cern i n t h e ev o l u t i o n o f P BP d ep en d en t t r an s p o rt ers. Th es e fe at u res m a y ex p l ai n t h e ad ap t i v e ad v an t a ge of i n co rp o rat i n g M BP i n t o t h e m al t o s e t ran s p o rt s ys t em, h o wev er i t s m ech an i s m o f act i o n rem ai n s m o re el u s i v e. Th e ab i l i t y o f M BP t o i n fl u en ce t h e act i v i t y o f c yt o s o l i c A BC s i s a co m p l ex s t u d y i n s i gn al t ran s d u ct i o n. 17

29 1.6 Unanswered Questions Concerning Maltose Transporter Activity: S t u d y o f t h e m al t o s e t ran s p o rt er h as b e en fo cu s ed l arg el y o n t h e cl o s ed fo r m o f M BP as t h e l i k el y e ffe ct o r o f t r an s p o rt er act i v at i o n. Th is i s b ecau s e h i gh l ev el s o f t ran s p o rt er act i v at i o n are o n l y s e en i n t h e p res e n ce o f m al t o s e, wh i ch i n du ces M BP t o ad op t t h e cl o s ed s t at e [ 5 7 ]. Ad d i t i o n al l y, l i g an d s t h at b i n d t o M BP wi t h o u t i nd uci n g d o m ai n cl o s u re a r e n o t t ran s p o rt ed b y t h e m al t o s e t ran s p o rt er [ 5 8 ]. Th e cl o s ed co n fo rm ati o n o f M BP t h ere fo re a p p ears t o p l a y a s i gn i fi can t ro l e i n t ran s p o rt er a ct i v at i o n an d i t wa s p res u m ed t h at t h e op en fo rm o f M BP rep res en t ed an i n a ct i v e s t at e. Ho wev e r, a l o n g s t an d i n g ob s e rv at i o n ch al l en g es t h i s u n d ers t an d i n g. W h i l e h i gh l ev el s o f ATP as e a ct i v at i o n are o n l y ob s erv ed i n t h e p res en ce o f M BP an d m al t o s e, u n l i gan d ed M BP s t i m u l at es a l o w l ev el o f ATP as e act i v i t y fro m t h e t ran s p o rt er. Th i s i s an u n u s u al res u l t as s t ru ct u re d et e rm i n at i o n b y b o t h cr ys t al l o gr ap h i c a nd s m al l -an gl e X-r a y s ca t t eri n g an al ys i s i n d i cat es t h at u n l i gan d ed M BP ad op t s a p ri m ari l y open co n fo rm at i o n [ 2 6 ]. Act i v at i o n b y u n l i gan d ed M BP h as b e e n ex p l ai n ed as ari s i n g fro m a s m al l fract i o n o f u n l i gan d ed M BP t h at t ran s i en t l y ad op t s a cl o s e d -l i k e co n fo rm at i o n wi t h ou t t h e s t ab i l i z i n g i n fl u en ce o f b o u n d m al t o s e [ 2 6 ]. S u ch a co n fo rm at i o n wo u l d b e h i gh l y u n s t a b l e [ 5 9 ]. Ho wev er, a n u m b er o f s t u d i es d o i n d i cat e t h at t h e u n l i g an d ed p ro t ei n can ad op t cl o s ed o r p art i al l y cl o s ed s t at es [ 6 0,6 1 ]. Th i s ex p l an at i o n h as b een d ub b ed t h e l o ck an d k e y h yp o t h es i s b ec au s e o f t h e i m p l i ci t as s u m p t i o n t h at s u rface c o m p l em en t ari t y b et ween t h e t ran s p o rt er an d cl o s e d M BP i s s u ffi ci en t t o act i v at e ATP as e. Th e l o ck an d k e y h yp o t h es i s i s i nd i cat i v e o f t h e gen eral v i ew o f t h e t ran s p o rt er at t h e i n i t i at i o n o f t h i s t hes i s, as i t i s gro u n d ed i n a b el i ef t h at t h e cl o s ed fo rm o f M BP i s t h e s o le act i v at o r o f ATP as e. 18

30 1.7 Research Goals and Hypothesis Th e p rev al en t t h eo ri es o f M al FGK 2 fu n ct i o n s u g g es t t h at t h e cl o s ed l i g an d - b o u n d co n fo rm at i o n o f M BP i s t h e p ri m ar y ef fe ct er o f A TP as e i n i t i at i o n. Ho wev er t h e as s o ci at i o n o f M BP wi t h t h e t ran s p o rt er at m u l t i p l e s t ages i n t ran s p o rt s u g ges t s t h at t h e ro l e o f M BP m a y b e m o re s op h i s t i cat ed ; wi t h b o t h co n fo rm at i o n s o f M BP an d t h e r el at i v e s t ab i l i t y o f t h o s e fo rm s p l a yi n g i n t e gr al ro l es i n r egu l at i n g ATP as e co up l e d t ran s p o rt. Bas ed o n i n fer en ce fro m p rev i o u s s t u d i es [ 4 4,5 7,6 2,6 3 ] an d an ex am i n at i o n o f t h e av ai l ab l e s t ru ct u r al i n fo r m at i o n [ 2 8,1 3,1 7], a h yp ot h et i cal m o d el o f m al t o s e t ran s p o rt e r act i v i t y h as b een d ev el op ed b y w h i ch t h e t ran s p o rt e r i s re g ul at ed t h ro u gh a s eri es o f co m p et i n g co n fo rm at i o n s [ 2 2 ]. Un d er t h i s m o d el, u n co up l ed ATP h yd ro l ys i s i s i n h i b i t ed b y a d es t ab i l i z at i o n o f t h o s e t ran s p o rt er co n fo rm at i o n s ad op t ed d u ri n g t h e c at al yt i c c yc l e. Th i s effe ct i v el y l i m i t s acces s t o ATP as e co m p et en t co n form at i o n s o f t h e t ran s p o rt er ( Fi g 1.4 ). Th e ro l e o f M BP i s t h en t o m o d u l at e t h e rel at i v e s t ab i l i t y o f t h es e co n fo rm at i o n s s o as t o m ed i at e i n t er-co n v e rs i o n b et we en t h em (Fi g 1.4 ). Th e t wo st ab l e fo rm s o f M BP, l i g an d ed -cl o s ed an d u n l i gan d ed -op en, wo u l d each b i n d an d s t ab i l i z e a d is t i n ct t ran s p o rt er co n fo rm at i o n. Fi rs t t h e b i n d i n g o f cl o s ed M BP a l l o ws co n v ers i o n o f t h e t r ans p o rt er r es t i n g s t at e i n t o t h e ATP as e cat al yt i c co n fo rm at i o n. Th e cat al yt i c s t at e i s t h en s t ab i l i z ed b y op en co n fo rm at i o n M BP t o i n i t i at e ATP h yd ro l ys i s i n res p o n s e t o t h e m o v em en t o f m al t o s e. Th e b i n d i n g p ro t ei n wo u l d t h u s fav o r o t h er wi s e h i gh -en e rg y i n t e rm ed i at e s t ep s i n tran s p o rt as a w a y t o ac c el erat e act i v i t y, i n a m an n er t h at s h ar es b ro ad s i m i l ari t y wi t h en z ym e c atal ys i s b y t ran s i t i o n s t at e s t ab i l i z at i o n [ 2 2]. Th i s t h es i s d es cri b es ef fo rt s t o t es t t h i s m o d el of ATP as e co up l i n g. Th e co n s erv ed d o m ai n s t ru ct u re o f t h e ABC t ran s p o rt er s up er fa m i l y m ak es t h e m al t o s e t ran s p o rt er an i d eal 19

31 Fig 1.4 Proposed Mechanism of Coupling in MalFGK 2 Th e m al t o s e t ran s p o rt e r u n d er go es a s eri es o f co n form at i o n al ch an g es d u ri n g t h e cat al yt i c c yc l e. Di f fer en ces i n t h e rel at i v e s t ab i l i t y o f t h es e co n fo rm at i o n s co u l d g a t e ATP h yd ro l ys i s b y b a rri n g a cces s t o t h e ATP as e cat al yt i c s t at e w h en m al t o s e i s n o t av ai l a b l e fo r t ran s p o rt. Un d er t h i s regu l at o r y s ch em e t h e t ran s p o rt e r co n fo rm at i o ns n o rm al l y b o u n d b y M BP wo u l d ex i s t as h i gh -en er g y i n t erm ed i at e s t h at req u i red s t ab i l i z at io n b y M BP. Fi rs t t h e b i n d i n g o f cl o s ed M BP en ab l es r ap i d co n v ers i o n o f t h e t ran s p o rt e r res t i n g s t at e i n t o t h e ATP as e cat al yt i c co n fo rm at i o n. Th e cat al yt i c s t at e i s t h en s t ab i l i z ed b y op en co n fo rm at i o n M BP t o i n i t i at e ATP h yd ro l ys i s i n res p o n s e t o t h e m o v em en t o f m al t o s e. 20

32 21

33 m o d el s ys t em t o i n v es t i g at e t ran s p o rt m e ch an i cs wi th rel ev an c e t o a s t a g g eri n g d i v ers i t y o f s ub s t rat es. Th e u l t i m at e go al o f t h i s l i n e o f rese ar ch i s t o d ev el op an u n d e r s t an d i n g o f A BC co up l ed t ran s p o rt m e ch a n i cs t h at wi l l en ab l e t h e r e-en gi n e eri n g o f t h es e s ys t em s wi t h m u t agen i c an d s m al l m o l ecu l e i n t er v en t i o n s t h at co u l d op t i mi z e i n d u s t ri al l y d es i rab l e t ran s p o rt p ro c es s es an d t reat t r an s p o rt er b as e d d i s eas es. 1.8 Experimental Methodology Th e fu l l m al t o s e t ran s p o r t er co m p l ex, wi t h b o u n d MBP, i s a rel at i v el y l ar g e co m p l ex o f k Da [ 1 7 ]. Th i s s iz e, as wel l as i t s m em b ran e em b ed d ed en v i ro n m en t, m ak es i t d i ffi cu l t t o p red i ct t h e e ffe ct o f t ar g e t ed m u t at i o n s wi t h i n t h e co m p l ex. Ho wev er as a s ub s et o f t h e co m p l ex, M BP h as b een m u ch b et t er ch a ra ct eri z ed i n t erm s o f i t s s t ru ct u re, co n fo rm a t i o n al ch an ges an d i n t er-d o m ai n i n t era ct i o n s. M BP i s al s o rel at i v el y s m al l, fu l l y s o l ub l e i n t h e aq u eo u s p h as e an d wel l k n o wn fo r i t s h i gh l y s t ab l e l o w-en e r g y fo l d. Fo r t h es e reas o n s M BP i s wi d el y u s ed i n fu s i o n p ro t ei n s t o au gm e n t t h e s o l ub i l i t y o f o t h e r p ro t ei n s. Th es e p rop ert i e s m ak e M BP an ap p eal i n g ex p eri m en t al t o o l, an d g reat l y s i m p l i f y th e en gi n eeri n g o f m u t an t s. Th e ex p eri m en t al ap p ro ach t ak en i n e ach c h ap t er o f t h i s t h es i s was t o en gi n eer t a r get ed m u t at i o n s wi t h i n M BP, ch ara ct eri z e t h ei r eff ect, an d t h en d et erm i n e wh at i n fl u en ce t h es e m o d i fi cat i o n s h av e h ad o n t h e ab i l i t y o f M BP t o a ct i v at e t h e t ran s p o rt e r ATP as e. P red i ct i n g an d ob s erv i n g t h e eff ect s o f M BP m u t at i o n s o n t ran s p o rt er act i v i t y i s a m e an s t o t est m o d el s o f t ran s p o rt er co up l i n g. M BP i s a cl as s i cal t yp e II b i n d i n g p ro t ei n, i n t h at i t co n s i s t s o f t wo l argel y i n d ep en d en t d o m ai n s co n n ect ed b y a f l ex i b l e h i n ge r e gi o n (Fi g 1.5 ) [ 2 6,2 7 ]. Each d o m ai n co n t ai n s a p o rt i o n o f t h e s ub s t rat e-b i n d i n g s i t e. Aft er i n i t ial b i n d i n g o f s ub s t rat e t o o n e o f t h es e d o m ai n s a s eri es o f i n cr eas i n gl y fav o rab l e i n t er act i o n s b et we en t h e s ub s t rat e an d p ro t e i n d raws t h e t wo 22

34 d o m ai n s cl o s ed, co m p l et i n g t h e fu l l b i n d i n g s i t e a nd p art i al l y o ccl u d i n g t h e s ub s t rat e wi t h i n t h e p ro t ei n [ 6 4 ]. Th i s i s o ft en refe rred t o as t h e V en u s Fl yt r ap m e ch an i s m f o r t h e wa y i n wh i ch t h e t wo d o m ai n s ap p ear t o cl o s e aro u n d an d gras p t h e b o u n d s ub s t rat e [ 6 5 ]. Th i s co n fo rm at i o n al ch a n ge i s n o rm al l y i n h i b i t ed b y a s t ru ct u re k n o wn as t h e b al an ci n g i n t erfac e. Lo c at ed op p o s i t e t h e s ub s t rat e-b i n d i n g s i t e, acro s s t h e h i n ge r e gi o n, t h e b al an ci n g i n t erfac e i s a cl e ft -l i k e i n t erfa ce b et w een t h e t wo d o m ai n s co n s i s t i n g o f h yd rop h ob i c res i d u es. Th es e res i d es p ack t o get h er an d ex cl u d e w at er w h en M BP i s i n t h e op en c o n fo rm at i o n (Fi g 1.5 A) b u t m u s t u n d er go s ep arat i o n an d h yd rat i o n, d i s fav o red b y en t rop y, w h en M BP re al i gn s i t s d o m ai n s d u ri n g cl o s u r e o f t h e m al t o s e-b i n d i n g s i t e (Fi g 1.5 B )[ 2 5,6 6 ]. W h en m al t o s e i s p res en t t h i s en er get i c co s t i s co u n t ered b y f av o rab l e i n t e r act i o n s b et ween M BP an d m al t o s e. It s h o u l d b e n o t ed t h at t h e b al an ci n g i n t erfa ce d o es n o t h o l d t h e p ro t ei n ri gi d l y i n a s i n g l e op en co n fo rm at i o n, b u t rat h e r s t ab i l i z es an en s em b l e o f cl o s el y rel at ed op en co n fo rm at i o n s wi t h rare an d s t o ch as t i c fo r a ys i n t o l es s s t ab l e cl o s ed s t at es. Th e s i gn i fi c an c e an d d e gr ee o f t h i s o s ci l l at i o n i s d eb at ed [ 6 0,6 1,5 9 ] an d i s ad d res s ed i n ch ap t ers 2 an d 4. Th e wel l -s t u d i ed n at u r e o f t h es e co n fo rm at i o n al c h an g es an d t h e i n t ri n s i c s t ab i l i t y o f t h e p ro t ei n m ak e M BP an ap p eal i n g t ar get fo r t h e en g i n eeri n g o f i n fo rm at i v e m u t an t s. Di s rup t i o n o f t h e M BP b al an ci n g i n t er f ace, ei t h er b y r em o v al o f fav o rab l e i n t era ct i o n s o r d i s rup t i o n o f s i d e- ch ai n p ack i n g, p u s h es t h e p ro t ei n t o ward s t h e cl o s ed co n fo rm at i o n, i n c rea s i n g t h e a ffi n i t y fo r m al t o s e [ 2 5,6 6 ]. S i m il arl y, gre at er s t ab i l i z at i o n o f o n e co m p et i n g co n fo rm at i o n o r al t erat i o n o f t h e s ub s t rat e-b i n d i n g s i t e can c au s e p r ed i cab l e s h i ft s i n t h e eq u i l i b ri u m b et ween op en an d cl o s ed co n fo rm at i o n s. Th es e fa ct o rs co m b i n e wi t h t h e s i gni fi c an t co n t ro l M BP ex ert s o v er ATP h yd ro l ys i s t o m ak e t h e b i n d i n g p ro t ei n an i d eal ex peri m en t al p rob e fo r t h e s t u d y o f t h e m al t o s e t ran s p o rt er. 23

35 Fig 1.5 The Substrate Binding Site and Balancing Interface Balance Conformational Changes in MBP. X-ra y cr ys t al s t ru ct u r es o f t h e m al t o s e b i n d i n g p ro t ei n are s h o wn i n t h e: (A ) Un l i gan d ed op en co n fo rm at i o n an d ( B) t h e m al t o s e b o u n d cl o s ed co n fo rm at i o n. Th e t wo d o m ai n s o f M BP (R ed an d M a g en t a) ro t at e wi t h res p e ct t o each o t h er fo l l o wi n g t h e b i n d i n g o f m al t o s e ( gre en ). Th i s d o m ai n ro t at i o n cau s es an op en i n g o f t h e h yd rop h ob i c b al an ci n g i n t e rf ace (b l u e) wh i l e i n d u ci n g t h e cl o s u re o f t h e s ub s t rat e b i n d i n g cl eft ( ye l l o w). P db r ef eren ce: 1 OM P [ 2 6],1 ANF [ 2 7 ] 24

36 25

37 1.9 Scope of Thesis Th e s eco n d ch ap t er o f t h i s t h es i s d et ai l s a car eful e x am i n at i o n o f t h e k i n et i cs o f ATP as e act i v at i o n b y m al t o s e s at u rat ed an d d ep l et ed M BP. Th e p u rp o s e o f t h i s i n v es t i gat i o n was t o t es t t h e p rev i o u s l y d i s cu s s ed l o ck an d k e y h yp o t h es i s, t h at s u rfac e co m p l em e n t ari t y wi t h cl o s ed M BP t ri g g ers t h e t ran s p o rt er ATP as e. A n o v el d e n at u rat i o n an d d i al ys i s a p p ro ach h as b een u n d ert ak en t o r em o v e al l co n t am i n at i n g m al t o s e f r o m p rep ar ed M BP. Th i s h as en ab l ed ob s erv at i o n o f t h e p r ev i o u s l y u n ex am i n ed k i n et i cs of ATP as e s t i m u l at i o n b y u n l i gan d ed M BP. Ou r fi n d i n gs i n d i c at e t h a t t h e M BP co n cen t r at i o n d ep en d en ce o f t h i s a ct i v a t i o n i s n o t co n s i s t en t wi t h cu rren t t h i n k i n g o n t h e s ub j ect. It i n s t ead su g ges t s t h at t h e op en co n fo rm at i o n o f M BP can d i rect l y b i n d an d act i v at e a rar e co n fo rm at i o n o f Mal FGK 2. Th i s s u g g es t s t h at m u l t i p l e fo rm s o f M BP m a y b e r es p o n s i b l e fo r t ran s p o rt e r a ct i v at i o n. Th e t h i rd ch ap t e r p res en t s fi n d i n gs u t i l i z i n g a n ov el s ub s t rat e-b i n d i n g m u t a n t o f M BP. Th i s m u t an t u n d ergo es c o n fo rm at i o n al ch an g es fr o m op en t o cl o s ed s i m i l ar t o wi l d t yp e M BP, b u t i s cap ab l e o f d o i n g s o i n res p o n s e t o t h e b i n d i n g o f s u cro s e as wel l was m al t o s e. P o i n t m u t at i o n s i n t h e s ub s t rat e-b i n d i n g s i t e cre at e fa v o rab l e i n t er act i o n s t h at en ab l e s u cro s e b i n d i n g t o co u n t er t h e op en i n g o f t h e b al an ci n g i n t er fac e. W e fi n d i n t h i s s t u d y t h at t h es e m u t at i o n s are s u ffi ci en t t o ad ap t ATP as e act i v at i o n i n t h e wi l d -t yp e t r an s p o rt e r t o a n o v el s ub s t rat e. Th es e fi n d i n gs d em o n s t rat e t h at t h e TM d o m ai n s d o n o t d i rect l y re co gn i z e t h ei r t ar get s ub s t rat e as a n eces s a r y s t ep i n act i v at i n g ATP h yd ro l ys i s. B as ed o n X-r a y s t ru ct u r e d et erm i n at i o n a m ech an i s m i s p rop o s ed b y wh i ch t h e t ran s p o rt e r c an i n d i rect l y t rack t h e p a s s ag e o f s ub s t rat e fro m M BP i n t o t h e t ran s l o cat i o n p at h wa y. In t h e fo u rt h ch ap t er ad d i t i o n al M BP co n fo rm at i o n al m u t an t s h av e b een p r ep ared. C ys t ei n e m u t at i o n s wer e i n t ro d u ced wi t h t h e i n t en t t o s t ab i l iz e t h e op en an d cl o s ed 26

38 co n fo rm at i o n s i n d i ffe ren t m u t an t s. C ro s s l i n k s b et ween t h es e res i d u es c rea t e co n fo rm at i o n al co n s t rai n t s t h at al t er t h e ab i l i t y t o i n t erco n v e rt b et ween cl o s ed an d op en s t a t es. Th i s h as en ab l ed t h e i n fl u en ce o f t h es e co n fo rm at i o n s o n t h e t ran s p ort er t o b e s ep arat el y i n v es t i gat ed. Th e e ffe ct o f t h es e m u t an t s o n wi l d - t yp e t r an s p o rt er h as b een ad d i t i o n al l y ex am i n ed af t er ex t ract i o n o f t h e co m p l ex fro m t h e m em b ran e b y a m i l d d et er g en t. Th is t reat m en t i s k n o wn t o cau s e s ub s t an t i al u n co up l i n g o f ATP as e a c t i v i t y; d i s rup t i n g t h e t ran sp o rt er s n o rm al re gu l at i o n. Th e s u s cep t i b i l i t y o f t h e t ran s p o rt e r t o act i v at i o n b y t h e co n s t rai n ed m u t an t b i n d i n g p ro t ei n s i s fo u n d t o d i ffer i n fo rm at i v el y, s u g g es t i n g an en e r get i c b arri e r a gai n s t u n co up l ed act i v at i o n t h at h as i t s o ri gi n s i n p ack i n g i n t e ract i o n s b et ween t h e TM s ub u n i t s an d t h e m em b ran e. 27

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43 Chapter 2 Stimulation of the Maltose Transporter ATPase by Unliganded Maltose Binding Protein 2.1 Introduction ATP Bi n d i n g C as s et t e ( ABC ) t ran s p o rt ers u s e t h e ch em i cal en e r g y o f AT P h yd ro l ys i s t o t ran s p o rt s o l u t es acro s s a m em b ran e. ABC t r an s p o rt ers c an b e d i v i d ed i n t o ex p o rt s ys t em s an d i m p o rt s ys t em s, an d a k e y q u es t i o n i n b o t h cas es i s t h e m ech an i s m b y wh i ch ATP b i n d i n g an d h yd ro l ys i s ar e re gu l at ed an d co up l ed t o s ub s t rat e tran s l o cat i o n. Fo r AB C ex p o rt s ys t em s, t h e s ub s t rat e i t s el f re gu l at es t h e ATP as e [ 1,2,3 ]. AB C i m p o rt s ys t em s, o n t h e o t h er h an d, i n cl u d e a p eri p h er al s ub s t rat e b i n d i n g p ro t ei n, an d t h e ATP a se act i v i t y i s r e gu l at ed b y i n t eract i o n s b et ween t h e b i n d i n g p ro t ei n an d t h e t ran s m em b ran e co m p o n en ts [ 4,5,6 ]. Th e E. coli m al t o s e t ran s p o rt er (M al F GK 2 ) i s a t ract ab l e an d w el l -s t u d i ed ABC i m p o rt s ys t em co n s i s t i n g o f t h e m em b ran e as s o ci at ed c o m p l ex M al FGK 2 an d p eri p h er al ex t racel l u l ar m al t o s e b i n d i n g p ro t ei n (M BP ). M al F an d M al G ar e i nt e gr al m em b r an e p ro t ei n s ; M al K 2 i s a d i m er o f A BC s ub u n i t s b o u n d t o M al FG o n t h e c yt op l as m i c s i d e o f t h e m e m b ran e. S t ru ct u res o f i s o l at ed M al K 2 h av e b e en s o l v ed an d fro m t h e s e i t i s k n o wn t h at AT P b i n d s i n t h e d i m er i n t erfac e an d p ro m o t es a t i gh t as s o ci at i o n o f t h e su b u n i t s, b ri n gi n g e ach AT P m o l ecu l e i n co n t a ct wi t h cat al yt i c res i d u es f ro m t h e op p o s i t e s ub u n i t [ 7]. On ce ATP h yd ro l ys i s t ak es p l ac e, t h e s ub u n i t s d i s s o ci at e, al l owi n g rel e as e o f ADP an d i no rgan i c p h o s p h at e [ 8 ]. In t h e i n t act M al FGK 2 co m p l ex, t h es e co n fo rm at i o n al ch an ges ar e co up l ed to ch an ges i n M al F G [ 9,1 0,1 1,1 2 ] s o t h at t h e m al t o s e b i n d i n g s i t e i n M al FG op en s al t ern at el y t o t h e p e ri p l as m o r c yt op l as m [ 1 2 ]. S t i m u l at i o n o f ATP h yd ro l ys i s b y t h e b i n d i n g p ro t ei n m u s t i n v o l v e co n fo rm at i o n al ch an ges t h at a re t r an s m i t t ed fro m t h e ex t racel l u l a r s u rfa ce t h ro u gh t h e t r a n s m em b ran e d o m ai n s t o t h e ATP b i n d i n g cas s et t es o n t h e c yt op l as m i c s i de. M BP an d o t h e r cl as s I o r II b i n d i n g 32

44 p ro t ei n s co n s i s t o f t wo d o m ai n s co n n ect ed b y a fl ex i b l e h i n ge, an d l i g an d b i n d i n g b ri n gs ab o u t a l arg e s t ru ct u ral ch an ge o f t h e p ro t ei n [ 1 3 ]. Th i s co n fo rm at i o n al ch a n ge, fro m t h e op en u n l i gan d ed s t ru ct u r e, t o t h e cl o s ed l i gan d ed fo r m, i s cri t i cal fo r t ran s p o r t s i n ce o n l y t h e l i gan d - b o u n d co n fo rm at i o n can fu l l y s t i m u l at e t h e m em b ran e ATP as e [ 4,1 4 ]. On t h i s b as i s, t h e l i gan d - i n d u ced co n fo rm at i o n al c h an g e s erv es t o en s u r e t h at fu t i l e c ycl es o f ATP h yd ro l ys i s ar e av o i d ed. Th i s p i ct u re i s co m p l i cat ed b y t h e en i gm at i c ob s e rvat i o n, i n b o t h t h e m al t o s e an d h i s t i d i n e t ran s p o rt s ys t em s, t h at ATP h yd ro l ys i s i s s t i mu l at ed, t o a l i m i t ed d eg ree, b y t h e unliganded b i n d i n g p ro t ei n [ 4,5 ]. W h i l e t h ere i s n o ob v i ou s p h ys i o l o gi c al fu n ct i o n fo r t h e ab i l i t y o f t h e u n l i gan d ed b i n d i n g p ro t e i n t o s t im u l at e t h e m em brane ATP as e, i t d o es rai s e q u es t i o n s ab o u t t h e m ech an i s m o f t h e s t i mu l at i o n. S p eci fi cal l y, i t i s no t cl ear wh at co n fo rm at i o n o f M BP i s res p o n s i b l e fo r t h e act i v at i o n o f t h e M al FG K 2 ATP as e i n t h e ab s en ce o f m al t o s e. On t h e o n e h an d, u n l i g an d ed M BP ex i s t s p red o m i n an t l y i n an op en co n fo rm at i o n i n s o l u t i o n [ 1 5,1 6 ], an d t h erefo r e t h e op en co n fo rm at i o n rep res en t s a l o gi cal can d i d at e fo r t h e act i v at i o n. In s up p o rt o f t h i s i d ea, d u ri n g m al t o s e t ran s p o rt an d at t h e p o i nt wh ere ATP i s p o i s ed fo r h yd ro l ys i s, t h e op en u n l i gan d ed fo rm i s fo u n d t i gh t l y b o u n d t o M al FGK 2 [ 1 1,1 7 ]. On t h e o t h er h an d, m al t o s e t ran s p o rt req u i res an i n i t i al i n t eract i o n wi t h cl o sed, m al t o s e-b o u n d M BP p ri o r t o op en i n g an d p ro g res s i o n t o t h e t ran s i t i o n s t at e fo r ATP h yd ro l ys i s. Th er efo r e, t h e a ct i v at i o n o f t h e M al FG K 2 ATP as e b y u n l i gan d ed M BP co u l d b e d u e t o a s m al l am o u n t o f a cl o s e d, u n l i gan d ed fo rm o f M BP wh i ch ex i s t s i n s o lu t i o n [ 1 8] ; t h e p res en ce o f s u ch a s o l u t i o n co n fo r m at i o n i s s up p o rt ed b y t h e cr ys t al l i z at i o n o f t w o o t h er b i n d i n g p ro t ei n s in a cl o s ed, u n l i gan d ed s t at e [ 1 9,2 0 ]. On t h i s b as i s, a cl o s ed u n l i gan d ed co n fo rm at i o n m i gh t affe ct t h e s am e ch an g es b ro u gh t ab o u t b y t h e cl o s ed liganded co n fo rm at i o n. Th i s m o d el fo r s t i m u l at i o n o f t h e ATP as e b y t h e b i n d i n g p ro t ei n 33

45 co u l d b e t erm ed l o c k -an d -k e y s i n c e i t i s s o l el y th e co n fo rm at i o n an d t h e s u rface co m p l em en t ari t y o f t h e b i n d i n g p ro t ei n t h at i s i m p ort an t fo r t h e i n t er act i o n. Ho wev e r, s ub s t rat e b i n d i n g aff ect s n o t o n l y t h e co nfo rm at i o n, b u t al s o th e s t ab i l i t y an d m o l ecu l ar d yn am i cs o f t h e b i n d i n g p ro t ei n. In t h e cas e o f M BP, i n t ri n s i c t ryp t op h an fl u o r es cen c e an d m o l ecu l ar d yn am i cs s i m u l at i o n s i n d i cat e t hat u nl i gan d ed M BP h as a s o m ewh at d yn am i c s t ru ct u re, wi t h t h e t wo do m ai n s m o v i n g rel at i v e t o each o t h er, i n co n t ras t t o t h e l i gan d -b o u n d fo rm i n wh i ch t h e rel at i v e p o s i t i o n s o f t h e d o m ai ns ap p ears t o b e fi x ed [ 2 1,2 2 ]. Fu rt h erm o re, t h e cl o s ed unliganded co n fo rm at i o n h as a m u ch h i g h er en er g y an d l o w er s t ab i l i t y t h an t h e cl o s ed, l i gan d ed fo rm d u e t o t h e ab s en ce o f s t ab i l i z i n g n o n-co v al en t b o n d s b et we en m al t o s e an d M BP [ 2 3 ]. Th u s, al t h o u gh a cl o s ed, u n l i g an d ed co n fo r m ati o n o f M BP co u l d, i n p ri n ci p l e, r es em b l e t h e cl o s ed l i gan d ed fo rm, i t wi l l b e m o re d yn am i c an d mu ch l es s s t ab l e. On t hi s b as i s, s ti m u l at i o n of t h e M al F GK 2 ATP as e b y a cl o s ed, u n l i g an d ed co n fo rm at i o n o f M BP i m p l i es t h at t h e co n fo rm at i o n al s t ab i l i t y o f t h e b i n d i n g p ro t ei n i s n o t cri t i cal fo r a p ro d u ct i v e i n t er act i o n, an d t h i s h as s i gn i fi c an t m ech an i s t i c i m p l i cat i o n s fo r h o w con fo rm at i o n al ch an ges are b ro u gh t ab o u t b y t h e i n t eract i o n b et w een M BP an d M al F GK 2. To ad d r es s t h e q u es t i o n o f wh et h er t h e cl o s ed, u n l i gan d ed fo rm o f t h e b i n d i n g p ro t ei n c an s t i m u l at e ATP h yd ro l ys i s b y t h e t ran s p o rt er, w e en gi n eer ed m al t o s e b i n d i n g p r o t ei n s o t h at t h e op en co n fo rm at i o n i s d es t ab i l i z ed rel at i v e t o t h e cl o s ed co n fo rm at i o n, an d as s a yed t h e ab i l i t y o f t h es e fo rm s o f t h e p ro t ei n t o act i v at e t h e m em b r a n e-b o u n d ATP as e. In ad d i t i o n, we h av e ca rri ed o u t a ca refu l an al ys i s o f t h e ab i l i t y o f u n l i g an d ed M BP t o s t i m u l at e t h e m em b ran e ATP as e, an d d em o n s t rat e t h at t h e ch aract eri s t i cs o f t h i s s t imu lat i o n are co m p l et el y d i fferen t fro m wh at i s ob s erv ed fo r l i gan d -b o u n d M BP. Th e res u l t s we h av e ob t ai n ed m ak e i t h i gh l y u n l i k el y t h at, i n 34

46 t h e ab s en ce o f m al t o s e, a cl o s ed co n fo rm at i o n o f M BP i s res p o n s i bl e fo r s t i m u l at i o n o f M al FGK 2, an d w e s u g g e s t i n s t ead t h at i t i s t h e m ore s t ab l e an d ab u n d an t op en co n fo rm at i o n. 35

47 2.2 Materials and Methods Bacterial strains and plasmids Th e b act eri al s t rai n HS was u s ed fo r ex p res s i o n fo r al l t ran s p o rt co m p l ex es, an d was o ri gi n al l y d eri v ed fro m t h e p ar en t al s t r ai n o f Escherichia coli K1 2 [ 2 4 ]. Th i s s t rai n co n t ai n s t h e malb1 0 1 al l el e, a d el et i o n o f t h e en t i re mal op ero n, an d t h e atp al l el e, a d el et i o n o f t h e gen e fo r F 1 -ATP as e. E. coli s t rai n HS [ 2 5] was u s ed fo r ex p res s i o n o f p eri p l as m i c M BP an d M BP -DM f ro m p LH1 [ 2 6 ] an d p LH1 -DM [ 2 7 ]. Th es e p l as m i d s co d e fo r M BP an d M BP -DM wi t h a p eri p l as m i c ex p ort t ag, u n d e r a M al T i n d u ci bl e p ro m o t er. HS d o es n o t p ro d u ce ch ro m o s o m al l y en co d ed M BP an d h as co n s t i t u t i v e ex p res s i o n o f t h e m a l t o s e op ero n act i v at o r, M al T t o d ri v e ex p res s i o n fro m p LH 1. In t h e M B P -DM m u t an t, res i d u es 1 7 2, 1 7 3, 1 7 5, an d are d el et ed, res u l t i n g i n a t ru n cat ed s u rf ac e l o o p, an d M an d Q3 2 5 ar e b o t h m u t at ed t o al an i n e. Th e p l as m i d p NTS K+ co n t ai n s malf an d malg gen es p r eced ed b y t h e IP T G-i n d u ci b l e p Tac p ro m o t er. p N TS K+ was p ro d u c ed b y d i g es t i n g t h e p l as m i d p C P 8 [ 2 4 ] wi t h Eco R 1 t o rem o v e t h e t et h e red M B P ex p res s i o n cas s et t e. S i n ce t h i s cas s et t e al s o co n t ai n ed t h e am p i ci l l i n res i s t an ce m ark e r, p Bl u es cri p t II S K + ( Ferm en t as ) was cl o n ed i n t o t h e Eco R 1 s i t e t o res t o re am p i ci l l i n res i s t an ce. p M R 1 1, wh i ch i s rep l i c at i v e-co m p at i b l e wi t h p NT S K+, h arb o rs t h e malk gen e p rec ed ed b y t h e p Tac p ro m o t er s eq u en c e, an d con t ai n s a ch l o ram p h en i co l res i s t an ce m ark er. p M al -9 6 W W co n t ai n s t h e co d i n g s eq u en ce fo r c yt op l as m i c M BP wi t h t h e b al an ci n g i n t erfac e m u t at i o n s A9 6 W an d I3 2 9 W [ 2 3 ]. Construction of hexahistidine-tagged MBP. To i n creas e t h e ef fi ci en c y o f p u ri fi cat i o n an d t h e yi el d o f M BP, t h e M BP co d i n g s eq u en ce was cl o n ed i n t o p P ro EX-HTa ( In v i t ro g en ) fo r i n t racel l u l ar ex p res s i o n o f a h ex ah i s t i d i n e-t ag ge d v ers i o n o f M BP co n n e ct ed b y a TE V p ro t eas e cl eav ab l e l i n k er. R es t ri ct i o n s i t es fo r cu t t i n g an d i n s ert i n g Eh e1 an d Hi n d III w er e i n t ro d u ced i n t o t h e p LH1 v ect o rs b y m u t ag en i c P C R, wi t h p ri m ers : 36

48 5 C GC C TC GGC TGGC GC C AAAATC GAAG -3 (fo rw ard ) an d 5 C GC C GC ATC C GGCATTTAAGC TTATT AC TTGGTGATAC G AG-3 (rev e rs e). Th e en t i re M BP co d i n g s eq u en c e was v e ri fi ed. Expression and purification of MBP Wild-Type MBP and MBP-DM. Al l ch ro m at o g rap h i c m ed i a w ere p u rc h as ed f ro m GE Heal t h ca re. P l as m i d s co n t ai n i n g wi l d -t yp e (p LH 1 ) o r m u t an t (p LH1 -DM ) male co d i n g r e gi o n s were t ran s fo rm ed i n t o E. coli s t rai n HS C u l t u res w ere g ro wn wi t h v i go ro u s s h ak i n g at 3 0 C i n LB b ro t h co n t ai n i n g µ g/ m L am p i ci l l i n fo r 1 6 t o 1 8 h. P eri p l as m i c pro t ei n s were ex t ract ed b y o s m o t i c s h o ck an d d i al yz ed a g ai n s t 5 0 m M Tri s -HC l, p H 8.5 i n p rep a rat i o n fo r an i o n ex ch an g e ch ro m at o g rap h y. Th e ex t ract w as ap p l i ed o nt o a 2.6 x 1 5 cm c o l u m n p ack ed wi t h Q - S ep h aro s e F as t Fl o w an d el u t ed wi t h a l i n ear grad i en t fro m 0 t o 1 M NaC l, wi t h a b as e b u ffer o f 5 0 m M Tri s -HC l, p H 8.5. Fra ct i o n s co n t ai n i n g M BP were p o o l e d an d d i al yz ed a gai n s t 5 0 m M Tri s -HC l, p H 8.5. Dep en d i n g o n t h e p u ri t y o f M B P con t ai n i n g fra ct i o n s at t h i s s t ep, a fu rt h er i o n - ex ch an g e s t ep u s i n g a M o n o -Q co l u m n was s o m et i m es neces s ar y b efo r e m o v i n g o n t o t h e n ex t s t ep. To eff ect fu rt h er p u ri fi c at i o n an d rem o v e co n t a m i nat i n g m al t o s e f ro m p rep ar at i o n s o f M BP, p ro t ei n fro m t h e i o n ex ch an ge co l u m n (s ) was d i l u t ed i n 5 0 m M Tri s -HC l, an d d i al yz ed ag ai n s t t wo ch an ges o f a fo l d ex ces s 2 M gu a n i di n e h yd ro ch l o ri d e. Gu an i d i n e h yd ro ch l o ri d e was t h en s l o wl y rem o v e d b y d i al ys i s a gai n s t a fo u r ch an ges o f a fo l d ex ces s o f 5 0 m M Tri s - HC l p H 8.5. Th e refo l d e d M BP was co n c en t rat ed b y i on -ex ch an ge ch ro m a t o grap h y u s i n g a 1 -m L Hi Trap Q co l u m n. C o n c en t rat ed p ro t ei n was l o ad ed o nt o a 2.6 x 6 0 cm co l u m n o f S up erd ex P rep Gr ad e g el fi l t rat i o n res i n, wh i ch h ad b e en e q ui l i b rat ed wi t h 2 0 m M Hep es, m M KC l, p H 7.4. Th e co l u m n was d ev el op ed wi t h t h i s s am e b u ffer at a fl o w r at e o f 0.5 m L/ m i n. Fra ct i o n s co n t ai n i n g p u re M BP were p o o l ed an d d i al ys ed a gai n st 2 ch an ges o f a fo l d ex ces s o f 5 0 m M 37

49 Tri s p H 8.0. M BP was co n cen t rat ed o n a 1 m L Hi Trap Q co l u m n, an d t h en d i al yz ed a gai n s t a fo l d ex ces s o f 5 0 m M Tri s -HC l p H 7.0, m M KC l, 1 0 m M M gc l 2. MBP-A96W/I329W. To ex p res s an d p u ri f y t h e c yt op l as m i c M BP -A9 6 W / I3 2 9W, HS cel l s wer e t ran s fo rm ed wi t h p M al -A9 6 W / I3 2 9 W, an d a s i n gl e co l o n y was u s ed t o i n o cu l at e m L LB -Am p. Th i s cu l t u r e was g ro wn wi t h v i go ro u s s h ak i n g t o an op t i c al d en s i t y o f 0.1 an d µ L was ad d ed t o 1 L o f LB -Am p. Th e cu l t u re w as gro wn t o an OD o f 0.4 an d ex p res s i o n of M BP A9 6 W / I3 2 9 W was i n d u ced b y ad d i t i o n o f µ g/ m L IP TG an d t h e cu l t u re g ro wn fo r a fu rt h er 6 h o u rs at 3 7 C. C el l s were rup t u red b y p ass a ge t h ro u gh a Fr en ch P res s u re cel l at 2 0,0 0 0 p.s.i, an d p u r e M BP w as ob t ai n ed b y an i o n -e x ch an ge an d g el fi l t rat i o n ch ro m at o gr ap h y as d es cri b ed ab o v e. Histidine-Tagged MBP. Fo r l at e r ex p eri m en t s i n v o l v i n g s t i m u l at i o n o f M al FGK 2 wi t h h i gh co n cen t r at i o n s o f M BP, wt M BP was ex p res s ed i n t racel l u l arl y i n a B L2 1 ( DE3 ) b ack gro u n d, as a fu s i o n p ro t ei n wi t h a TEV p ro t eas e cl e av ab l e h ex ah is t i d i n e t ag. Aft er g ro wt h, i n d u ct i o n, an d h arv es t i n g (as d es cri b ed fo r M BP -A9 6 W / I3 2 9 W, ab o v e) t h e cel l p el l et was res u s p en d ed i n 5 0 m L o f Ni 2 + -NTA b u ffe r (2 0 m M Hep es, m M KC l, 1 0 m M i m i d az o l e, 1 0 % gl yce ro l, p H 8.0 ) an d s up p l em en t ed wi t h 1 m M P M S F b efo re l ys i s b y 3 p as s es t h ro u gh a Fren ch p res s u r e cel l at 2 0,0 0 0 p s i. C el l d eb ri s was p el l et ed at 1 0 0,0 0 0 g an d t h e s up ern at an t ap p l i ed t o Ni 2 + -l o ad ed C h el at i n g S ep h aro s e F F an d p ro t ei n s el u t ed u s i n g an i m i d az o l e g rad i en t fro m 1 0 t o m M. Hi gh p u ri t y fra ct i o n s co n t ai n i n g M BP wer e p o o l ed and a 1 : 2 0 m as s rat i o o f TEV p ro t eas e was ad d ed ; t h e s o l u t i o n was d i al yz ed o v ern i gh t at 4 C ag ai n s t 4 L o f Ni 2 + -NTA b u ff er t o rem o v e ex ces s i m i d az o l e. Th e TEV p ro t eas e t r eat ed p r o t ei n was l o ad ed o n t o cl e a n Ni 2 + -NTA res i n an d M BP, wi t h t h e h i s t i d i n e t ag r em o v ed, was co l l ect ed fro m t h e fl o w t h ro u gh. Th e cl eav ed M BP was d i al yz ed o v ern i gh t a gai n s t 4 L o f 5 0 m M Tri s -HC l, p H 8.5, i n p rep ar at i o n fo r an i o n ex ch an ge ch ro m at o grap h y. 38

50 Denaturation and refolding of MBP. Fra ct i o n s fro m t h e an i o n ex ch an g e co l u m n c o n t ai n i n g p u r e M BP w ere p o o l ed an d d en at u red b y o v e rn i gh t d i al ys i s a g ai n s t a 5 - fo l d ex ces s o f 6 M gu an i d i n e-hc l. Th e d en at u r ed p ro t ei n was s ub s eq u en t l y d i al yz ed a g ai n s t 5 ch an g es o f a 1 0 -fo l d ex ces s o f 6 M gu an i d i n e-hc l at 4 º C, res u l t i n g i n a l i gan d d i l u t i o n o f 5 0 0, fo l d; 5 mm EDTA was in cl u d ed i n t h e fi n al 2 d i al ys i s b u ffe rs. P ro t ei n was refo l d ed b y d i al ys i s ag ai n s t 1 M gu an i d i n e- HC l, 5 m M EDTA, an d t h en d i al yz ed t wi c e a gai n s t a 5 0 -fo l d ex ces s o f 5 0 m M Tri s -HC l p H 8.5. No t e t h at t h e p rop e r r efo l d i n g an d fu n ct i o n o f al l M BP p rep arat i o n s was t es t ed b y fl u o res cen ce t i t rat i o n s w i t h m al t o s e an d / o r m al t o t ri o s e: t h e r efo l d ed M BP p rep arat i o n s t yp i c al l y yi el d ed a h i gh er d e gre e o f fl u o r es cen ce q u en c h i n g du e t o co m p l et e r em o v al o f l i gan d. To m ai n t ai n co n s i s t en c y b et ween ATP as e as s a ys i n b o t h t h e ab s en ce an d p r es en ce o f m al t o s e, al l M BP p rep a rat i o n s wer e s ub j ect ed t o t h e s am e u n f o l d in g an d r efo l d i n g p ro c ed u re. Preparation of Proteoliposomes Over-expression of MalFGK 2. HS cel l s were co -t r an s fo rm e d wi t h p l as m i d s carr yi n g t h e m al F an d m al G g e n es (p NTS K+), an d t h e m al K gen e (p M R 1 1 ). A s i n gl e co l o n y was i n o cu l at ed i n t o m L o f LB (co n t ai n i n g µ g/ m L am p i ci l l i n, an d 5 0 µ g/ m L ch l o ram p h en i co l ), an d gr o wn t o a OD o f 0.1 ; µ L o f t h i s cu l t u re was ad d ed t o e ach l i t e r o f t h e s am e m ed i a, an d ce l l s were g ro wn wi t h v i go ro u s s h ak i n g at 3 7 C, t o an OD 6 00 o f 0.4; ex p res s i o n o f M al FGK 2 was i n d u ced b y t h e ad d i t i o n o f µ g/ m L IP TG an d gro wn fo r 8 h o u rs at 3 7 C. Th e c el l p el l et f ro m each l i t er w as was h ed t wi ce wi t h 5 0 m L o f 2 0 m M Hep es p H 7.0, an d cru d e m em b ran es w ere p r ep ar ed as fo l l o ws : each gr am o f w as h ed c el l s was re-s u s p en d ed i n 1 0 m L o f 2 0 m M H ep e s 7.0, 5 m M M gc l 2, wi t h 1 0 % gl yce ro l, an d p as s ed t wi ce t h ro u gh a Fren ch p r es s u re cel l at 1 5,0 0 0 p s i. C el l d eb ri s w as rem o v ed b y cen t ri fu g at i o n at 7,0 0 0 g fo r

51 m i n u t es. Th e s up ern at a n t was t h en s ub j ect ed t o u l t racen t ri fu gat i o n at 1 0 0,0 0 0 g t o reco v er m em b ran es. M em b ran es were was h ed t wi ce i n 1 0 m M M OPS p H 7.5, 5 m M M gc l 2, mm s u cro s e, an d res u s p en d e d i n 1 0 m M M OP S p H 7.5, 5 m M M gc l 2, m M s u cro s e, wi t h 1 0 % m et h an o l. Solubilization of membrane protein. W as h ed m em b ran e v es i cl es w er e reco v e red b y cen t ri fu gat i o n at 1 0 0,0 0 0 g an d re-s u s p en d ed at a p ro t ei n co n cen t rat i o n o f 5 m g/ m L i n 5 0 m M Tri s -HC l, p H 7.0, 1.2 % β -o ct yl gl u co s i d e, an d 1 0 m M M gc l 2. Th i s s o l u tio n was k ep t o n i ce wi t h o ccas i o n al a gi t at i o n fo r 6 0 m i n u t es, an d was s ub s equ en t l y s ub j e ct ed t o u l t racen t ri fu g at i o n at 1 0 0,0 0 0 g fo r 4 5 m i n u t es t o p el l et t h e i ns o l ub l e m at eri al. Th e rem ai n i n g s up ern at an t co n t ai n i n g t h e s o l ub i l i z ed p ro t ei n fract i o n was k ep t at 4 C u nt i l u s e. Preparation of Proteoliposomes (PLS). C ru d e E. coli p h o s p h o l i p i d s (Av an t i P o l ar Li p i d s ) were d i s s o l v ed at a co n cen t rat i o n o f 5 0 m g/ m L i n chl o ro fo rm. Th e ch l o ro fo rm was r em o v ed u s i n g a ro t a r y ev ap o rat o r, l eav i n g a l i p i d fi l m o n t h e ro u n d b o t t o m fl as k, an d t h e l i p i d s were re - h yd r at ed wi t h 5 0 m M T r i s -HC l, p H 7.0, an d 1 m M DTT at a co n c en t rat i o n o f 5 0 m g/ m L. Li p i d al i q u o t s o f µ L w e re q u i ck - fro z en, an d s t o red u nd e r a r go n at 8 0 C u n t i l u s e. Li p i d s u s p en s i o n s were t h awed an d s o n i cat ed t o cl ari t y u si n g a m i cro t i p s o n i cat o r b efo re t h e n ex t s t ep. To reco n s t i t u t e t h e i n t act t ran s p o rt er i n t o P LS, µ L o f s o l ub i l i z ed t ran s p o rt er (5 m g/ m L) was m i x ed wit h µ L (5 0 m g/ m L) o f s o n i cat ed l i p i d s, an d k ep t o n i ce 3 0 mi n u t es. P LS we re t h en fo rm ed u s i n g t h e d et e r g en t d i l u t i o n p ro ced u re [2 8 ] as fo l l o ws : t h e p ro tei n / l i p i d s o l ut i o n was d i l u t ed s l o wl y u s i n g a p eri s t al t i c p u m p wi t h 5 0 m M Tri s -HC l, p H 7.0, an d 1 m M DTT t o a v o l u m e o f 2 0 m L. P LS were r eco v e red b y u l t ra c en t ri fu gat i o n at 1 0 0,0 0 0 g fo r 4 5 m i n u t es. P LS were t h en was h ed o n ce u s i n g t h i s s am e b u ff er an d re-s u s p en d ed i n µ L. To t al p ro t ei n d et erm i n at i o n was d o n e u s i n g t h e Lo wr y m et h o d. T yp ical l y P LS s o l u t i o n s were at a fi n al p ro t ei n co n cen t r at i o n o f ap p ro x i m at el y 2 m g/ m L. 40

52 Assay of ATPase activity P LS wer e ad d ed t o yi el d a fi n al co n cen t r at i o n o f 0.1 m g/ m L i n 5 0 m M Tr i s -HC l p H 8.0, m M KC l, 1 0 mm M gc l 2 i n a t o t al v ol u m e o f µ L. P u ri fi ed M BP was ad d ed wi t h an d wi t h o u t 5 m M m al t o s e. Th e re act i o n w as s t art ed b y th e ad d i t i o n o f µ L o f 5 0 m M T ri s -HC l, 8 m M ATP, 1 0 m M M gc l 2, p H 8.0. Al i q o ut s o f µ L wer e rem o v ed a t 0, 5, 1 0, 2 0 an d 3 0 m i n u t e t i m e i n t erv al s an d ad d ed t o µ L o f 1 0 % S DS t o s t op t h e re act i o n an d d i s rup t t h e P LS. Th e am o u n t o f free p h o s p h at e l i b erat ed d u ri n g t h e react i o n was t h en m o n i t o red b y ad d i n g µ L co l o r re a gen t (1 0 m M a m m o n i u m m o l yb d at e an d 1 m M FeS O 4 i n 1 N H 2 S O 4 ) an d m eas u ri n g t h e ab s o rb an c e at n m. S tan d ard s o l u t i o n s o f p o t as s i u m p h o s p h at e were m e as u red u s i n g t h e s am e rea gen t s t o p ro d u ce a s t a n d ard p h o s p h at e cu rv e. To co n s erv e b i n d i n g p ro t ei n s an d l i p o s o m es, we al s o u s ed a 5 -fo l d s cal ed - d o wn v ers i o n o f t h e as s ay wi t h a fi n al v o l u m e o f µ L r at h er t h an t h e 1.5 m L d es cri b ed ab o v e. In t h i s cas e, P LS an d M BP were m i x ed i n a v o l um e o f µ L, an d t h e react i o n was i n i t i at ed w i t h 1 8 µ L o f 8 0 m M A TP, 5 0 m M Tri s -HC l, m M KC l, p H 8.0. Al i q u o t s o f 6 0 µ L w ere r em o v ed at 0, 5, 1 0, 2 0, an d 3 0 m i n u t es an d m ix ed wi t h 3 0 µ L 1 0 % S DS, fo l l o wed b y 5 0 µ L o f co l o r rea g en t. CD Spectroscopy Th erm al d en at u rat i o n o f wi l d -t yp e an d m u t an t M BP m ol ecu l es was m o n i t o red u s i n g a J ASC O J A s p ect rop o l ari m et er. P ro t ei n s we re d i al yz ed a g ai n s t 1 0 m M s o d i u m p h o s p h at e, p H 7.4, wi t h o r wi t h o u t µ M m al t o s e. Bu ff er s c an s wer e a cq u i red u n d er s i m i l ar co n d i t i o n s. A t h erm al m el t was carri ed o u t wi t h t h e refo l d ed p ro tei n s i n a 0.1 cm p at h len gt h q u a rt z cu v et t e b y m o n i t o ri n g t h e C D s i gn a l at n m. Th e s am p l e was h eat ed fr o m 2 0 t o 95 C wi t h a h eat i n g rat e o f 7 5 C / h u s i n g a J AS CO P TC W I P el t i er d ev i ce. A b an d wi d t h o f 0.2 n m an d a res p o n s e 41

53 t i m e o f 4 s we re u s ed. T ran s i t i o n p o i n t s wer e d et erm i n ed b y l i n ear ex t rap o l at i o n o f t h e p l at eau re gi o n s u s i n g t h e fo l l o wi n g eq u at i o n [ 2 9 ] : = ( ) ( ) (1) In t h i s ex p res s i o n x i s t h e t em p erat u r e i n d e gre es Kel v i n ; y n an d y u refe r t o t h e m o l ar el l i p t i ci t y o f t h e fo l d ed a n d u n fo l d ed p l at eau s, r es p ect i v el y; m n an d m u are t h e s l op es o f t h e p l at eau s ; R i s t h e u n i v ers al g as co n s t an t i n u n i t s o f k cal m o l -1 K -1 ; T m is t h e m el t i n g t em p er at u re i n d egr ees K el v i n ; an d H m i s t h e en t h al p y o f u n f o l d i n g at T m i n k cal m o l -1. Th erm al d en at u rat i o n o f M BP i s l ar gel y i rr ev ers i b l e an d t h erefo re t h e fi t t ed T m an d H m v al u es l ack q u an t i t at i v e t h erm o d yn a m i c s i gn i fi can ce, b u t a re u sefu l fo r a s em i -q u an t i t at i v e co m p ari s o n o f t h erm al d en at u r at i o n i n t h e p res en ce an d ab s en ce o f m al t o s e. Small Angle X-ray Scattering S AXS ex p eri m en t s were carri ed o u t at Bi o C AT b eam l i ne ID1 8 at t h e A d v an ced P h o t o n S o u rce (Ar go n n e Il l i n o i s, U.S.A.) es s en t i al l y as d e s cri b ed p rev i o u s l y [ 3 0 ]. R ad i i o f g yrat i o n w ere cal cu l at ed wi t h t h e p ro gr am GNOM [ 3 1 ] an d CRYSOL [ 3 2 ] was u s ed fo r m at ch i n g c r ys t al s t ru ct u res t o S AXS d at a. Modelling of MBP-Dependent ATPase Kinetics S t ead y s t at e an d m as s b al an c e eq u at i o n s we r e cr eat ed b as ed o n t h e k i n et i c m o d el s i l l u s t rat ed i n Fi gu re 2.4. In t h es e s ch em es, BP o and BP c rep r es en t o p en an d cl o s ed M BP, res p ect i v el y, an d M rep res en t s t h e m em b ran e c o m p l ex s u ch t h at BP om an d BP cm are t h e co rres p o n d i n g co m p l ex e s b et ween M BP an d M al FGK 2. Fo r each o f t h e k i n et i c m o d el s, t h e 42

54 eq u at i o n s wer e s o l v ed an al yt i cal l y, p ro v i d i n g t h e st ead y s t at e co n cen t rat i o n o f t h e act i v e co m p l ex i n t erm s o f t h e t o t al am o u n t o f b i n d i n g p rot ei n an d m em b ran e co m p l ex, p l u s t h e rat e co n s t an t s. Th e rat e co n s t an t s are o n l y r el at i v e v al u es : t h e y wer e i n i t i al l y s e t at 1 an d t h en adj u s t ed t o refl ect t h e ob s erv ed p r op ert i es o f t h e s ys t em. Fo r t h e c as e wh e re o n l y t h e cl o s ed fo rm o f M B P s t i m u l at es ATP h yd ro l ys i s (Fi gu re 4 A), eq u at i o n s 2 t o 6 were u s e d : [M ]=[M]+[BPoM]+[BPcM] (2) d[bp ]=[BPo]+[BPc] (3) [BPo]= [BPc] (4) [ ] [ ] =0= [BPo][M]+[BPcM]( + ) [BPoM]( + ) (5) =0= [BPc][M]+ [BPoM] [BPcM]( + + ) (6) Fo r s i m p l i ci t y, t h e eq u at i o n s i n co rp o rat e t h e fo l l owi n g as s u m p t i o n s : fi rs t, t h e m em b ran e - b o u n d b i n d i n g p ro t ei n rep res en t s a rel at i v el y s m al l p rop o rt i o n o f t h e t o t al, an d t h e refo re m em b ran e-b o u n d fo rm s are n o t i n cl u d ed i n t h e m as s bal an ce eq u at i o n f o r t o t al b i n d i n g p ro t ei n (eq u at i o n 3 ); s e co n d, t h e op en an d cl o s ed fo rm s o f t h e fr ee b i n d i n g p ro t ei n are as s u m ed t o b e i n eq u i l i b ri u m (eq u at i o n 4 ). Fo r t h e cas e t h at i n cl u d es t h e p res en ce o f an a l t ern at e co n fo rm at i o n o f t h e m em b ran e co m p l ex (d en o t ed b y M * ) an d act i v at i o n b y op en M BP (Fi gu re 4 B ), eq u at i o n s 6 t o 1 0 were u s ed. Here w e as s u m ed t h at t h e p rop o rt i o n o f t h e act i v at ed fo rm o f t h e m em b ran e co m p l ex (M * an d BP o M * ) was rel at i v el y s m al l, an d was t h e refo re n o t i n cl u d ed i n t h e m as s b al an ce eq u at i o n fo r t h e m em b ran e co m p l ex (eq u at i o n 7 ). Fu rt h e rm o re, al l o f t h e b i n d i n g p ro t ei n i s as s u m ed t o b e i n t h e op en, fre e fo rm ( eq u at i o n 8 ). 43

55 [M ]=[M]+[BPoM] (7) [BP ]=[BPo] (8) [ ] [ ] [ ] =0= [M]+ [BPoM ] [M ] [M ][BPo] (9) =0= [BPo][M]+[BPoM ]( + ) [BPoM]( + ) (10) =0= [BPo][M ]+ [BPoM] [BPoM ]( + + ) (11) 44

56 2.3 Results Stimulation of the MalFGK 2 ATPase by MBP I i n v es t i g at ed t h e ro l es o f M BP an d m al t o s e i n s t i m u l at i o n o f t h e M al F GK 2 ATP as e u s i n g a p ro t eo l i p o s o m e (P LS ) s ys t em. M al F GK 2 was o v er-ex p res s ed an d P LS were f o rm ed fro m cel l m em b ran es b y a d et er g e n t d i l u t i o n p ro ced u r e. Af t er d et e r gen t d i l u t i o n, M al FGK 2 wi l l b e p res en t i n b o t h o ri en t at i o n s i n t h e P LS b i l a ye r. A h i gh co ncen t rat i o n o f M g 2 + was u s ed t o p ro m o t e m em b ran e fu s i o n an d m i x i n g; t h erefo r e, fi x ed, cl o s ed v es i cl es d o n o t ex i st i n t h is s ys t em, al l o wi n g fo r b i -d i r ect i o n al m al t o s e t ran s p o rt [ 3 3 ]. Im p o rt an t l y, t h e s am e P LS p rep arat i o n can b e reco n s t i t u t ed wi t h v ar yi n g co n cen t r at i o n s o f M BP o r M BP m u t an t s, t h ereb y co n t ro l l i n g fo r d i fferen ces i n t h e am o u n t an d s p eci fi c act i v i t y o f M al FGK 2 i n t h e m em b ra n es. Th e p res en ce o f M al F, M al G, an d M al K i n t h e P LS was co n fi rm ed b y W es t ern b l o t t i n g (n o t s h o wn ). Th e P LS o n t h ei r o wn h ad a b as al ATP as e t h at rep r es en t s u n co up l ed act i v i t y f ro m M al FGK 2 an d / o r o t h er s o l ub l e o r m em b ran e -b o u n d ATP as es. R e gard i n g t h e p r ep ar at i o n o f M BP, we fo u n d t h at wh en M BP i s p u ri fi ed fro m a p eri p l as m i c ex t ract, i t co -p u ri fi es wi t h a co n t am i n at i n g ATP as e; i n ad d i t i o n, M BP t en d s t o ret ai n s o m e m al t o s e, ev en a ft er s ev e ral p u ri fi cat i o n s t ep s. C o n t a m i n at i o n o f M BP wi t h an ATP as e an d / o r m al t o s e can co n fo u n d t h e ATP as e m eas u rem en t s an d p recl u d e ac cu rat e co m p ari s on s b et ween t h e e ffect o f u n l i g an d ed an d m al t o s e-b o u n d M BP o n t h e M al FGK 2 ATP as e. I ad d res s ed t h es e p rob l em s b y i n t ra cel l u l ar ex p res s i o n o f M BP as a h ex a-h i s t i d i n e t ag ged fu s i o n p ro t ei n. Aft er i n i t i al p u ri fi cat i o n b y Ni NTA ch ro m at o gr ap h y a n d cl eav a g e o f t h e t a g, l eav i n g o n l y t wo ex t ra res i d u es, gl yc i n e an d al an i n e, at t h e N-t e rm i n u s, M BP w as p u ri fi ed b y i o n-ex ch an g e ch ro m at o gr ap h y yi el d i n g a s i n gl e b an d b y S DS -P AGE wi t h n o s i gn i fi can t co n t am i n at i n g ATP as e act i v i t y. To en s u r e t h at i t was t o t al l y f ree o f m al t o d ex tri n s, t h e M BP was fu l l y d en at u r ed, d i al yz ed ex h au s t i v el y a gai n s t 6 M gu an i d i n e -HC l, an d t h en refo l d ed. P rop er r efo l d i n g an d fu n ct i o n o f M BP p rep ar at i o n s was 45

57 co n fi rm ed b y fl u o r es cen ce t i t rat i o n wi t h m al t o s e and / o r m al t o t ri o s e. Un l i gan d ed M BP p r ep ar ed i n t h i s m an n er was u s ed f o r al l as s a ys, wi t h o r wi t h o u t m al t o s e. Un l i gan d ed M BP (2 µ M ) s t i m u l at ed t h e ATP as e act i v i t y b y ap p ro x i m at el y 5 0 % at 2 0 C an d % at 3 7 C (Fi gu re 2.1 ). As d i s cu s s ed, t h e M BP p rep arat i o n t h at was ad d ed t o t h e P LS h ad n o s i gn i fi c an t ATP as e act i v i t y o n i t s o wn, an d h ad b e en fu l l y d e n at u red, d i al yz ed, an d refo l d ed t o en s u re t h at ab s o l u t el y n o m al t o s e w as pres en t. Th e refo re t h e s t i m u l at i o n was d u e s o l el y t o an i n t era ct i o n b et ween u n l i gan d ed M B P an d M al FGK 2. Th es e res u l t s, i n cl u d i n g t h e ab i l i t y o f u n l i gan d ed M BP t o s t i m ul at e M al FGK 2, are s i m i l ar t o wh at was ob s erv ed p rev i o u s l y fo r t h e m al t o s e t r an s p o r t s ys t em [ 4 ], an d a s i gn i fi can t s t i m u l at i o n b y t h e u n l i gan d ed b i n d i n g p ro t ei n h as al s o b een d o c u m en t ed fo r t h e h i s t i d i n e t ran s p o rt er [ 5 ]. Concentration dependence of MBP-mediated MalFGK 2 stimulation To fu rt h er ch ar act e ri z e t h e act i v at i o n o f t h e M a l FGK 2 ATP as e b y b o t h u n l i gan d ed an d l i gan d ed M BP, I s t u d i ed t h e ef fect o f ch an gi n g t h e M BP co n c en t rat i o n at b o t h 2 0 C an d 3 7 C. In t h e p res en ce o f m al t o s e, i n creas es i n M BP co n ce n t rat i o n l ed t o al m o s t p r op o rt i o n al i n cre as es i n ATP as e act i v i t y t h at b e gan t o r each a p l at eau at 1 0 µ M, p res u m ab l y as m em b ran e-b o u n d M al FGK 2 b ec am e s at u r a t ed wi t h m al t o s e-m BP. In t h i s r es p ect t h e s ys t e m res em b l es a s i n gl e s ub s t rat e en z ym e, wh e re M al FGK 2 i s t h e en z ym e an d M BP -m al t o s e i s t h e s ub s t rat e. The ATP as e rat e v e rs u s M BP co n cen t rat i o n d at a w ere fi t t o a s im p l e M i ch ael i s -M en t e n eq u at i o n, yi el d i n g a K M o f ap p ro x im at el y 1 5 µ M (d efi n ed as t h e co n c en t rat io n o f M BP -m al t o s e t h at yi el d s o n e h al f V M AX ) at b o t h 2 0 C an d 3 7 C, an d V M AX v al u es o f an d n m o l m g -1 m i n -1 at 2 0 C an d 3 7 C res p ect i v el y (T ab l e 2.1 an d Fi gu r e 2.2 ). 46

58 Fig 2.1 Stimulation of the MalFGK 2 ATPase by Unliganded MBP ATP h yd ro l s i s was m eas u red fo r p ro t eo l i p o s o m es o n t h ei r o wn (s o l i d gre y b a rs ) as wel l as wi t h 2 µ M M BP (s t i p p l ed b ars ) at 2 0 C an d 3 7 C. Th e ad d i t i o n o f M BP res u l t ed i n an 5 0 % i n creas e i n ATP as e act i v i t y at 2 0 C, an d a % i n creas e at 3 7 C. S h o wn are t h e m ean ± S.D.; a p ai red t -t es t was u s ed t o cal cu l at e t h e l i k el i h o o d t h at t h e d i ffer en c es aro s e b y ch an c e. 47

59 48

60 Fig 2.2 Kinetic Analysis of MBP-MalFGK 2 ATPase Stimulation in the Presence of Maltose Th e ab i l i t y o f wi l d -t yp e M BP t o s t i m u l at e t h e M al FGK 2 ATP as e, at M BP co n cen t r at i o n s fro m 2 t o µ M, was m eas u red i n t h e p res en ce o f 5 m M m al t o s e at (A) 2 0 C, fi l l ed ci rcl es, an d (B) 3 7 C, cro s s es. Th e s o l i d cu rv es s h o w t h e n o n -l i n ear l e as t -s q u ar es fi t s t o t h e M i ch ael i s - M en t en eq u at i o n, wh i ch yi el d ed K M v al u es (d efi n ed as t h e co n cen t r at i o n o f M BP t h at yi el d s h al f m ax i m al ATP as e) o f ap p ro x i m at el y 1 5 µ M an d V M AX v al u es o f an d n m o l m i n -1 m g -1 at 2 0 C an d 3 7 C, res p ect i v el y (T ab l e 1 ). Th e b as al rat e o f act i v i t y h as b een s ub t ract ed fro m t h es e d at a. No t e t h at al l M BP p rep arat i o n s i n cl u d ed an u n fo l d i n g an d re fo l d i n g s t ep, as d es c ri b ed i n M et h o d s, t o co m p l et el y r em o v e l i g an d. 49

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62 Th e s am e ex p eri m en t s w ere c arri ed o u t wi t h M B P t h at was co m p l et el y fr ee o f m al t o s e. At 2 0 C, ad d i t i o n o f u n l i ga n d ed M BP at co n cen t r at i o n s o f 2 µ M an d 2 0 µ M p ro d u ced ex act l y t h e s am e m o d es t i n creas e i n ATP as e act i v i t y ( Fi gu re 2.3A). Th i s i s i n co n t ras t t o t h e s i t u at i o n fo r m al t o s e-b o u n d M BP, i n wh i ch a s i m i l ar fo l d i n creas e i n co n cen t r at i o n p ro d u ced cl o s e t o a fo l d i n creas e i n a ct i v i t y (Fi gu re 2.2 A ). In c reas i n g u n l i g an d ed M BP co n c en t rat i o n fro m 1 t o µ M p ro d u ced n o s i gn i fi can t i n creas e i n t h e s t im u l ati o n o f M al FGK 2 ATP as e act i v i t y ( Fi gu re 2.3 B). W h en t h es e d at a were fi t t o a M i ch ael i s - M en ten eq u at i o n, t h e cal cu l at ed K M was 0.6 µ M, o v er 2 0 -fo l d l o w er t h an t h e K M fo r l i gan d ed M BP. W e al s o car ri ed o u t t h e as s a ys at 3 7 C wh ere t h e s t i m u l at i o n b y u n l i g a n d ed M BP w as gre at er, a l l owi n g u s t o ob t ai n r at e d at a at co n cen t r at i o n s o f u n l i gan d ed M BP l o w er t h an 1 µ M. At 3 7 C, i n creas i n g t h e u n l i gan d ed M BP co n c en t rat i o n fro m 0.1 t h ro u gh t o µ M p ro d u ced o n l y a m o d es t i n creas e i n t h e act i v at i o n o f M al FGK 2, wi t h a cal cu l at ed K M o f 0.8 µ M (Fi gu re 2.2 C an d T ab l e 2.1 ), al m o s t 2 0 -fo l d l o wer t h an t h e K M ob t ai n ed fo r m al t o s e-b o u n d M BP at 3 7 C. Kinetic models for stimulation of the MalFGK 2 ATPase by unliganded MBP Un l i gan d ed M BP m u s t s t i m u l at e t h e M al FGK 2 ATP as e t h ro u gh ei t h er i t s cl o s ed o r op en co n fo rm at i o n. To i n v es t i gat e i n m o r e d et ai l t h e ex pect ed M BP co n c en t rat i o n d ep en d en ce fo r t h es e t wo p o s s i b i l i t i es, we m o d el e d t h e k i n et i cs o f t h e act i v at i o n p ro ces s. A p o t en t i al co m p l i cat i n g f act o r i s t h e p as s i v e (i.e. n o n -a ct iv at i n g) i n t er act i o n b et w een u n l i g an d ed M BP an d M al FG K 2 wh i ch was fi rs t p o s t u l at ed o n a t h e o ret i cal b as i s an d t h en o b s erv ed ex p eri m en t al l y [ 3 4,3 5 ]. W e i n co rp o rat e d t h i s i nt eract i o n i n t o two m o d el s fo r t h e s t i mu l at i o n o f t h e M al FGK 2 ATP as e b y u n l i gan d ed M BP. Th e rel at i v e a ffi n i t y o f t h e op en co n fo rm at i o n o f M BP fo r t h e t ran s p o rt er i s ap p ro x i m at el y 3 -fo l d l es s t h an t h e c l o s ed fo rm ( Fi gu re 2.4 A an d Tab l e 2.2 ; k -1 /k 1 = 5 0, k -2 /k 2 = 1 5 ), wh i ch i s b as ed o n m at h em at i cal m o d el i n g o f t h e s ys t em a s wel l as t h e m eas u red affi n i t i es o f op en an d cl o s ed M BP fo r t h e i s o l at ed M al F P 2 l o op [ 1 6,3 5,3 6 ]. 51

63 Fig 2.3 Kinetic analysis of MBP-MalFGK 2 ATPase stimulation in the absence of maltose (A) In t h e ab s en c e o f m al t o s e, M BP p ro d u ces a s m al l b u t s i gn i fi can t s t im u l at i o n o f t h e M al FGK 2 ATP as e t h at i s ex act l y t h e s am e fo r 2 µ M an d 2 0 µ M co n cen t rat i o n s o f M BP at 2 0 C. Th e b ars s h o w t h e m e an ± S.D. fo r 6 t o 9 d et er m i n at i o n s i n each c as e. ( B) At 2 0 C an d i n t h e ab s en ce o f m al t o s e, i n cr eas es i n M BP co n cen t rat i o n fro m 1 t o µ M pro d u ced n o s i gn i fi c an t i n creas e i n M al FGK 2 ATP as e act i v i t y (fi l l ed ci rcl es ). No t e t h at act i v it i es b el o w 1 µ M M BP co n cen t r at i o n co u l d n o t b e rel i ab l y d et erm i n ed at 20 C. (C ) At 3 7 C an d i n t h e ab s en ce o f m al t o s e, t h e h i gh e r a ct i v i t y l ev el s al l o wed m ea s u rem en t o f t h e M al FG K 2 ATP as e a ct i v i t y at M BP co n c en t rat i o n s b el o w 1 µ M (cro s s es ). Th e d at a i n p an el s B an d C yi e l d ed K M v al u es o f l es s t h an 1 µ M an d V M AX v al u es o f 5 an d 5 0 n m o l m i n -1 m g -1 at 2 0 C an d 3 7 C, res p ect i v el y. Fo r t h e an al ys i s i n p an el s B an d C, t h e b as al ATP as e o f t h e p ro t eo l i p o s o m es (i n t h e ab s en c e o f an y M BP ) was s ub t ract e d fro m al l m eas u rem en t s. 52

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65 Table 2.1: Kinetic Parameters for Activation of MalFGK2 by Liganded and Unliganded MBP Conditions a K M (µm) V MAX (nmol min -1 mg -1 ) M al t o s e-b o u n d, 2 0 C (9.4 t o ) b ( t o ) Un l i gan d ed, 2 0 C (0 t o 1.8 ) 4.9 (3.7 t o 6.1 ) M al t o s e-b o u n d, 3 7 C (8.1 t o ) ( t o ) Un l i gan d ed, 3 7 C (0.1 7 t o 1.4 ) 5 0 (4 3 t o 5 8 ) a Th e b as al ATP as e a ct i v i t y m e as u red i n t h e ab s en ce o f M BP wa s s ub t ract ed fro m al l m eas u rem en t s. b Val u es i n p aren t h es es i n d i cat e t h e 9 5 % co n fi d en c e in t erv al, as p ro v i d ed b y t h e P ri s m s o ft ware. 54

66 In t h e fi rs t m o d el (Fi gu re 2.4 A), M BP al t ern at e s bet ween op en an d cl o s ed co n fo rm at i o n s wi t h b o t h fo rm s b i n d i n g t o M al FGK 2, b u t o n l y t h e cl o s ed fo rm s t i m u l at i n g ATP h yd ro l ysi s. To t es t t h e m o d el, we u s ed i t t o p red i ct wh at wo u l d b e ob s erv ed i n t h e p res en ce o f s at u rat i n g m al t o s e. Fo r t h i s, M BP wi l l b e p r es en t o n l y i n t h e cl o s ed, l i gan d ed fo rm, wh i ch c an b e ac co m p l i s h ed i n t h e m o d el b y m ak i n g t h e rat e co n s t an t s fo r t h e cl o sed t o op en t ran s i t i o n o f M BP (k -3 an d k -4 ) v er y s m al l (Fi gu r e 2.4 A an d Tab l e 2.2 ). W i t h t hi s set o f rat e co n s t an t s, t h e s ys t em p ro d u ces t h e ob s erv ed K M o f 1 5 µ M (Fi gu re 2.5 A); f ro m t h e m o d el i n g, we ob t ai n a re l at i v e co n cen t r at i o n o f t h e a ct i v e s p eci es (i n t h i s cas e, l i g an d ed M BP b ou n d t o M al F GK 2 ), wh i c h can v ar y f ro m 0 t o 1. A fact o r w as ap p l i ed t o ob t ai n a V ma x cl o s e t o t h e n m o l m i n -1 mg -1 t h at was ob s erv ed ex p eri m en t al l y. Th e s i t u a t i o n i n t h e ab s en c e o f m a lt o s e i s s i m i l ar, ex cep t t h at k -3 an d k -4 are m ad e l arg er t o refl e ct t h e s h i ft t o ward s t h e op en co n fo r m at i o n o f b i n d i n g p ro t ei n (Tab l e 2.2 ); t h e v al u e o f 1 0 was ch o s en fo r b o t h k -3 an d k -4 s o t h at t h e rat e o f ATP h yd ro l ys i s at 1 o r 2 µ M u n li gan d ed M BP co m p ared w el l wi t h wh at was ob s erv ed ex p eri m en t al l y. If t h e s ys t e m op erat ed t h ro u gh t h i s m ech an i s m i n t h e ab s en c e o f m al t o s e, t h en t h e ap p aren t K M fo r M BP wo u l d b e j u s t o v er 4 0 µ M, an d t h ere wo u l d b e a fo l d i n creas e i n t h e M al F GK 2 ATP as e act i v i t y as t h e M BP co n cen t r at i o n was rai s ed fro m 1 µ M t o 2 5 µ M (Fi gu re 2.5 B). W e at t em p t ed t o im p ro v e t h e ag re em en t wi t h ex p eri m en t al d at a b y u s i n g d i ff eren t p aram et er s e t s, b u t fo u n d t h at t h e o n ly w a y t o l o w er t h e K M fo r M BP was t o i n c reas e t h e af fi n i t y o f t h e i n t e ract i o n b et ween M al F G K 2 an d op en M BP (i.e. d ecre as e k -1 ) s u ch t h at i t i s at l eas t 2 0 -fo l d h i gh er t h an t h e affi n i t y o f t h e i n t era ct i o n b et ween M al FGK 2 an d cl o s ed M BP. S u ch p aram et ers a r e at o d d s wi t h t h e rel ati v el y we ak ab i l i t y o f u n l i gan d ed M BP t o i n h i b i t t ran s p o rt b y l i ga n d ed M BP [ 3 5 ] as wel l as d i r ect b i n d i n g m eas u rem en t s b et w een M BP an d t h e i s o l at ed P 2 l o op of M al F [ 3 6 ]. Th e al t ern at i v e m ech an i s m i n wh i ch t h e op en co n fo rmat i o n o f M BP s t i m u l at es t h e M al FGK 2 ATP as e p ro v i d es a b et t er fi t t o t h e ob s erv ed d at a. In t h i s m o d el (Fi gu re 2.4 B an d T ab l e 55

67 2.2 fo r p aram et e r v al u es ), o n l y op en u n l i gan d ed M BP i s p res en t an d i t h as t h e s am e p as s i v e i n t eract i o n wi t h M al F G K 2 as i n t h e p rev i o u s m o d el. Ho wev e r, t h er e i s a s econ d fo rm o f M al FGK 2, M *, wh i ch i s ab l e t o b i n d op en u n l i gan d ed M BP wi t h h i gh affi n i t y (n o t e t h e l o w v al u e o f k -2 i n Tab l e 2.2 ), p ro d u ci n g t h e t ran s i t i o n s t at e co m pl ex, l ead i n g t o ATP h yd ro l ys i s an d re gen e rat i o n o f M al FGK 2 t o i t s res t i n g co n fo rm at i o n. In ad d i t i o n t o t h e lo w rat e o f d i s s o ci at i o n o f t h e t r an s i t i o n s t at e co m p l ex, t h ere i s a l o w rate o f fo rm at i o n o f M *, wh i ch l i m i t s t h e m ax i m u m rat e o f cat al ys i s. Th e l o w rat e co n s t an t s fo r t h e fo rm at i o n o f M * (k 3 an d k 4 ), as w el l as t h e l o w rat e co n s t an t fo r d i s s o ci a t i o n o f t h e t ran s i t i o n s tat e co m p l ex (k -2 ) l e ad t o b eh av i o u r t h at i s cl o s e t o wh at was ob s erv ed, n am el y a K M o f ~1 µ M an d l o w V M AX. Th e K M can b e l o wer ed fu rt h er, t o s ub -m i cro m o l ar l ev el s, b y s l o wi n g t h e rat e o f co n v ers i o n o f M * t o M (d ecre as i n g co n s t an t s k- 3 an d k- 4 ; Tab l e 2.2 an d Fi gu r e 2.5 B ). In s u m m ar y, u n l i gan d ed M BP s t i m u l at es t h e M al FGK 2 ATP as e wi t h a K M b el o w 1 µ M, an d t h e n at u re o f t h e s t i m u l at i o n i s n o t co n s i s t en t wi t h a m ech an i s m i n wh i ch a m i n o r s p eci es ad op t s a cl o s ed, u n l i gan d ed co n fo rm at i o n t o m i m i c th e e ffe ct o f cl o s ed, l i gan d ed M BP. In s t ead, t h e s t i m u l at i o n i s co n s i s t en t wi t h a s m al l am o u n t of M al FGK 2 c ro s s i n g an en er get i c b ar ri er t o ad op t an al t ern at e co n fo rm at i o n t o wh i ch op en M BP bi n d s wi t h h i gh af fi n i t y, l e ad i n g t o A TP h yd ro l ys i s i n t h e ab s en ce o f m al t o s e. Conformational engineering of MBP To fu rt h er ad d res s t h e ro l es o f t h e op en an d cl o s ed co n fo rm at i o n s o f M BP, we u s ed co n fo rm at i o n al l y- en gi n eered M BP m o l ecu l es i n wh i ch t h e op en co n f o rm at i o n i s s el ect i v el y d es t ab i l i z ed rel at i v e t o t h e cl o s ed co n fo rm at i o n. S el ect i v e d es t ab i l i z at i o n o f t h e op en co n fo rm at i o n wi l l s h i ft t h e op en -cl o s ed eq u i l i b r i u m o f u n l i gan d ed M B P t o ward s t h e cl o s ed co n fo rm at i o n. On t h i s b a s i s, i f i t i s t h e cl o s ed con fo rm at i o n o f u n l i gan d ed M BP t h at s t i m u l at es 56

68 Fig 2.4 Kinetic Models for Stimulation of MalFGK 2 ATPase by Open and Closed MBP (A) S t i m u l at i o n b y cl o s ed M BP. Bo t h op en an d cl o s ed fo rm s o f M BP (BP o an d BP c, res p ect i v el y) i n t e ract wi t h M al FGK 2 b u t o n l y t h e cl o s ed fo rm s t i m u l at es ATP h yd ro l ys i s. In t h i s m o d el, t h e m ech an i s m o f ATP as e s t i m u l at i o n i s t h e sam e fo r b o t h u n l i g an d ed an d l i gan d ed M BP : t h e cl o s ed co n fo rm at i o n i n t eract s wi t h M al FGK 2 l ead i n g t o ATP h yd ro l ys i s. In t h e ab s en ce o f m al t o s e, t h e rel at i v el y l o w rat e o f ATP h yd ro l ys i s i s d u e t o t h e l i m i ti n g co n cen t rat i o n o f cl o s ed, u n l i gan d ed M BP. (B) S t i m u l at i o n b y op en M BP. Th e st i m u l at i on i s d u e t o a s eco n d co n fo rm at i o n o f M al F G K 2 (M * ) t h at i s s ep arat ed fro m i t s m o re p red o m i n an t res t i n g o r gro u n d s t at e (M ) b y an en er g et i c b arri er. Op en u n l i g an d e d M BP b i n d s wit h h i gh affi n i t y t o t h i s fo rm o f M al FGK 2 wh i ch l ead s d i rect l y t o fo rm at i o n o f t h e t ran s i t i on s t at e fo r ATP h yd ro l ys i s. In t h i s s ys t em, t h e l o w rat e o f ATP h yd ro l ys i s i n t h e a b s ence o f m al t o s e i s d u e t o t h e s l o w rat e o f co n v ers i o n b et w een M a n d M *. R el at i v e v al u es f o r t he rat e co n s t an t s ar e p ro v i d ed i n Tab l e

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70 Tab l e 2.2 : R at e C o n s t an t s fo r Ki n et i c M o d el s P aram et er S t i m u l at i o n b y C l o s ed M BP M al t o s e S at u rat ed C l o s ed Un l i gan d ed S t i m u l at i o n b y Op en Un l i gan d ed M BP k k k k k k k k k c a t

71 Fig 2.5 Expected rate curves for MalFGK 2 stimulation by MBP Th e k i n et i c m o d el s i n F i gu re 4 al o n g wi t h r at e co n st an t s i n Tab l e 2 w e re u s ed t o d eri v e cu rv es fo r t h e r at e o f ATP h yd ro l ys i s as a fu n ct i o n o f M BP co n cen t r at i o n. P an el s A an d B rep res en t t h e s am e grap h s, b u t wi t h ex p an d ed ax es in P an el B. In t h e p res en c e o f s at u rat i n g m al t o s e (s o l i d cu rv e), wh ere al l t h e b i n d i n g p ro t e in i s i n a cl o s ed co n fo rm at i o n (acco m p l i s h ed i n t h e m o d el b y m ak i n g t h e cl o s ed t o op en rat e co n s t ant s v er y s m al l ) t h e s ys t em s h o ws a K M fo r M BP o f 1 5 µ M, co r res p o n d i n g t o wh at w as ob s e rv ed ex p eri m en t al l y. In t h e ab s en ce o f m al t o s e, t h e m o d el wh er e t h e cl o s ed, u n l i gan d ed fo rm i s res po n s i b l e fo r t h e s t i m u l at i o n l ead s t o a K M o f j u s t o v er 4 0 µ M (l o n g d as h es ) an d a s i gn i fi c an t i n creas e i n act i v i t y as t h e co n cen t r at i o n o f M BP i s rai s ed i n t h e regi o n fr o m 1 t o 2 5 µ M. Act i v at ion b y op en u n l i gan d ed M BP (s h o rt d as h es ), as i l l u s t rat ed i n Fi gu r e 4 B a n d o u t l i n ed i n t h e t ex t, yi el d s res u l t s t h at ar e s i m i l ar t o wh at i s ob s erv ed ex p eri m en t al l y. 60

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73 M al FGK 2, t h en t h e s h i ft i n t h e op en -cl o s ed eq u i l ib ri u m s h ou l d res u l t i n a gr eat e r s t i m u l at i o n of M al FGK 2 i n t h e ab s en ce o f m al t o s e. S el ect i v e d es t ab i l i z at i o n o f t h e op en co n fo rm at i o n was a cco m p l i s h ed b y al t eri n g res i d u es i n a re gi o n we h av e cal l ed t h e b al an ci n g i n t erf ac e. M BP i s co m p o s ed o f t wo gl ob u l ar d o m ai n s co n n ect ed b y a fl ex i b l e h i n ge: t h e m al t o s e b i n d in g cl eft i s o n o n e s i d e o f t h e h i n ge, an d t h e b al an ci n g i n t er fac e i s o n t h e op p o s i t e s i d e, wh ere i t b al an ces o r co u n t eract s cl o s i n g o f t h e m al t o s e-b i n d i n g cl eft. O n e o f t h e co n fo rm at i o n al l y-en gi n eer ed M BP m o l ecu l es, M BP -DM, h ad b een m u t at ed t o r em o v e fav o r ab l e i n t e ract i o n s i n t he b al an ci n g i n t e rfa ce t h at s t ab i l i z e t h e op en co n fo rm at i o n [ 2 7 ]. C r ys t al s t ru ct u res an d s m al l an gl e X-r a y s c at t eri n g (S AXS ) o f M BP -DM i n d i cat ed t h at i t ad op t s ex act l y t h e s am e op en an d cl o s ed co n fo rm at i o n s as wi l d -t yp e M BP ; h o wev er, i t s m al t o s e a ffi n i t y i s ap p ro x i m at el y t i m es h i gh er t h an wi l d -t yp e d u e t o s el e ct i v e d es t ab i l i z at i o n o f t h e op en, u n l i gan d ed co n fo rm at i on. Ot h er gro up s h av e d es t ab i l i z ed t h e op en co n f o rm at i on b y r ep l ac em e n t o f b al an ci n g i n t erfac e r es i d u es wi t h b u l k i er s ub s t i t u en t s [ 2 3,3 7]. On e s u ch m u t an t, M BP -A9 6 W / I3 2 9 W, h as an av e ra g e s o l u t i o n co n f o rm at i o n co rr es p o n d i n g t o a d o m ai n cl o s u r e o f 2 8 [ 2 3 ], wh i ch i s cl o s e t o t h e 3 5 d o m ai n cl o s u r e i n m al t o s e-b o u n d M BP. W e u s ed S AXS t o fu rt h er v e ri f y t h e p ert u rb ed s o l u t i o n co n fo rm at i o n o f M BP -A9 6 W / I3 2 9 W. Ad d i t i o n of m al t o s e t o l i gan d - fre e M BP - A9 6 W / I3 2 9 W res u l t ed i n a b arel y d et ect ab l e ch an ge in t h e rad i u s o f g yr at i o n fro m Å fo r t h e u n l i gan d ed p ro t ei n, t o 22.1 Å fo r t h e m al t o s e-b o u n d p ro t ei n (Fi gu re 2.6 A). W h en co m p ared t o wi l d -t yp e M BP, t h e s o lu t i o n co n fo rm at i o n o f M BP -A9 6W / I3 2 9 W m o s t cl o s el y r es em b l es t h e cl o s ed m al t o s e-b o u n d s t r u ct u re o f M BP (Fi gu r e 2.6 B). As wi t h M BP -DM, t h e affi n i t y o f M BP - A9 6 W / I3 2 9 W fo r m al t o s e i s m u ch h i gh er t h an t h at o f wi l d -t yp e M BP, co n s i s t en t wi t h s el ect i v e d es t ab i l i z at i o n o f t h e op en co n fo rm at i o n. On t h i s bas i s, an d as s u m i n g t h a t t h e wi l d -t yp e p ro t ei n 62

74 ex i s t s t o s om e d egre e i n a cl o s ed, u n l i gan d ed co n form at i o n i n s o l u t i o n, b ot h o f t h e m u t an t M BP s are ex p ect ed t o s am p l e a cl o s ed u n l i gan d ed co n fo r m at i o n m o re freq u en t l y t h an wi l d -t yp e M BP. Th e s el e ct i v e d es t ab i l i zat i o n o f t h e op en, u n l i gan d ed co n fo rm at i o n s o f M BP -DM an d M BP -A9 6 W / I3 2 9 W c an b e s een fro m t h erm al d e n at u rat i on cu rv es ( Fi gu r e 2.7 ): i n t h e p r es en c e o f m al t o s e t h e T m v al u e s fo r wi l d -t yp e M BP, M BP -DM, an d M BP -A9 6 W / I3 2 9 W w ere s i m i l ar (6 5 C fo r wi l d -t yp e v ers u s ap p ro x i m at el y 6 4 C f o r t h e t wo m u t an t s ) wh ereas i n t h e ab s en c e o f m al t o s e t h e T m v al u es fo r M BP -DM an d M BP -A 9 6 W / I3 2 9 W wer e b o t h s i gn i fi can t l y d ec reas ed (t o 5 3 C ) co m p ared t o u n l i gan d ed wi l d -t yp e M B P (6 3 C ). Th e m u t at i o n s i n M BP -DM an d M BP - A9 6 W / I3 2 9 W are n o t d i rect l y i n v o l v ed i n l i gan d b i n di n g; fu rt h erm o re, s i n ce t h e y are o n t h e op p o s i t e s i d e o f t h e p ro t ei n fro m wh ere M BP an d M al FGK 2 i n t eract, t h e y wi l l n o t d i rect l y i n t erfer e wi t h b i n d i n g t o M al FGK 2 [ 1 1,2 5,2 6 ] an d t h e m u t at i o n s s h o u l d n o t aff ect t h e s t ru ct u r e o f an y cl o s ed, u n l i gan d ed co n fo rm at i o n s t h at m a y ex i s t i n s o l u t i o n. A p ro d u ct i v e i n t er act i o n b et ween t h es e m u t an t s a n d M al FGK 2 i s d em o n s t rat ed b y t h e fact t h at t h e y b o t h s up p o rt gro wt h o n m al t o s e m i n i m al m ed i a i n an M BP -d efi ci en t s t rai n (n o t s h o wn ). Fo r t h e p u rp o s es o f t h es e ex p eri m en t s, t h erefo re, t h e cl o s ed, m al t o s e-b o u n d fo rm s o f M BP -DM an d M BP -A9 6 W / I3 2 9 W are i d en t i c al t o wi l d -t yp e, b u t t h e op en co n fo rm at io n s h av e b een d es t ab i l i z ed. To ex p l o re t h e i m p o rt a n ce o f t h e op en an d cl o s ed co n fo rm at i o n s o f M BP i n m al t o s e t ran s p o rt, w e co m p ared t h e ab i l i t y o f wi l d -t yp e M BP an d M BP -DM t o s t i m u l at e t h e M al F GK 2 ATP as e i n b o t h t h e p res en ce an d ab s en ce o f m a l t o s e (Fi gu re 2.8 ). In t h e ab s en c e o f m al t o s e, M BP -DM s t i m u l at ed A TP h yd ro l ys i s t o t h e s a m e d e gre e as wi l d -t yp e. Th erefo re, s h i ft i n g t h e op en -cl o s ed eq u i l i b ri u m o f M BP t o wa rd s t h e cl o s ed co n fo rm at i o n d o es n o t en h an c e t h e ab i l i t y o f M BP t o s t i m u l at e M al F GK 2 i n t h e ab s en c e o f m al t o s e, as wo u l d b e ex p ect ed i f i t wer e t h e cl o s ed co n fo rm at i o n t h at i s res p o n s i b l e fo r t h e s t i m u l at i on. S u rp ri s i n gl y, i n t h e p re s en ce o f m al t o s e 63

75 Fig 2.6 Unliganded MBP-A96W/I329W is in a Closed Conformation (A) S m al l -an gl e X-r a y s cat t eri n g (S AXS ) fro m M BP -A9 6 W / I3 2 9 W was m eas u red i n t h e ab s en ce (t h i ck g re y cu rv e) an d p res en ce (t h i n b l ack cu rv e ) o f m al t o s e. Th e i n s et gr ap h s h o ws t h e d i fferen ce b et we en t h e t wo cu rv es, ex p res s ed as a per cen t a g e o f t h e t o t al s i gn al. Th e t wo cu rv es are al m o s t i d en t i cal, i n d i cat i n g n o ob s e rv ab l e e ff ect o f m al t o s e o n t h e s o l u t i o n co n fo rm at i o n o f t h e p ro t ei n. (B) Th e S AXS d at a fro m u n l i gan d e d M BP -A9 6 W / I3 2 9 W (t h i ck gr e y cu rv e ) was m at ch ed t o t h e op en s t r u ct u re o f M BP (P D B ID 1 OM P [ 4 3 ] ; d as h ed cu rv e) an d t h e cl o s ed, l i gan d -b o u n d s t ru ct u re (P DB ID 1 AN F [ 4 4 ] ; s o l i d cu rv e). Th e i n s et s h o ws t h e d i ffe ren c e b et w een t h e m o d el s an d t h e s o l u ti o n co n fo rm at i o n o f u n l igand ed M BP -A9 6 W / I3 2 9 W as a p ercen t a ge o f t h e t o t al s i gn al. Th e cl o s ed co n fo rm at i o n o f M BP i s a b et t er m o d el fo r t h e s o l u t i o n co n fo rm at i o n o f u n l i gan d ed M BP -A9 6 W / I3 2 9 W. 64

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77 Fig 2.7 Stability of Conformationally Engineered MBP Molecules C o n fo rm at i o n al l y en gi n e ered M BP m o l ecu l es, M BP -DM an d M BP -A9 6 W / I3 2 9 W, wer e p ro d u ced b y m ak i n g c h a n ges i n t h e b al an ci n g i n t erfa ce t h at co n t ro l s op e n i n g an d cl o s i n g o f t h e m al t o s e b i n d i n g cl e ft [ 2 0,2 1 ]. Th e t h erm al s t ab i l i ti es o f M BP -DM (s h o rt d as h es ) an d M BP - A9 6 W / I3 2 9 W (l o n g/ s h o r t d as h es ) were co m p ared t o wi l d -t yp e M BP (s o l i d ) i n t h e (A) ab s en ce o f m al t o s e, an d (B) p r es en ce o f µ M m al t o s e. Th e s am p l es wer e h eat e d at 7 5 K p er h o u r an d m ean res i d u e el l i p t i ci t y ( θ ) at n m was m eas u red ev er y 4 s eco n d s ( gr e y d at a p o i n t s ; s q u ares fo r wi l d -t yp e M BP, ci rc l es fo r M BP -DM, an d i n v ert ed t ri an gl es fo r M BP -A9 6 W / I3 2 9 W ). Th e cu rv es ar e n o n -l i n ear l e as t -s q u ares fi t s o f t h e d ata t o an eq u at i o n d es cri b i n g t h erm o d yn am i c t ran s i t i o n s i n t erm s o f t h e t wo p l at e au re gi o n s (s e e M et h o d s ). Th e b al an ci n g i n t er fa ce m u t at i o n s h av e d ec reas ed t h e s t ab i l i t y o f t h e u n l i gan d ed co n fo rm at i o n, b u t h av e n o s i gn i fi c an t eff ect o n t h e s t ab i l i t y o f t h e m al t o s e-b o u n d cl o s ed co n fo rm at i o n. 66

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79 M BP -DM was s i gn i fi can t l y i m p ai r ed i n i t s ab i l it y t o s t im u l at e M al FGK 2 co m p ared t o wi l d -t yp e M BP. Th i s was an u n exp ect ed res u l t b ec au s e t h e m u t at i o n s o n l y aff ect t h e s t ab i l i t y, an d n o t t h e s t ru ct u re, o f t h e op en c o n fo rm at i o n. Op en M B P fo rm s a t i gh t t ran s i t i o n s t at e co m p l ex wi th M al FGK 2 [ 1 1,1 7 ] an d t hes e k i n et i cs res u l t s s h o w t h at t h e bal an ci n g i n t erf a ce o f M BP co n t ri b u t es t o ATP h yd ro l ys i s, p rob a b l y b y en h an ci n g t h e s t ab i l i t y o f t h e t ran s i t i o n s t at e co m p l ex. Th e res u l t s ob t ai n ed wi t h M BP -DM we re rei n fo rc ed b y ex p eri m en t s wi t h M BP - A9 6 W / I3 2 9 W (Fi gu re 2.8 ). In t h e ab s en ce o f m al t o s e, s t i m ul at i o n of M al FGK 2 b y M BP - A9 6 W / I3 2 9 W was n o t s i gn i fi can t, an d i n fact t h e m u tat i o n s h av e d ecreas ed an d p o s s i b l y ab ro gat ed i t s ab i l i t y t o s t i m u l at e M al FGK 2 i n t h e ab s en ce o f m al t o s e. Gi v en t h at u n l i g an d ed M BP -A9 6 W / I3 2 9 W ad o p t s an av er a ge co n fo rm at i o n t h at i s al m o s t cl o s ed, t h e i n ab i l i t y o f u n l i gan d ed M BP - A9 6 W / I3 2 9 W t o s t i m u l at e M a l FGK 2 i s n o t co n s i s t en t wi t h t h e i d ea t h at t h e cl o s ed co n fo rm at i o n al o n e i s res p o n s i b l e fo r s t i m u lat i o n i n t h e ab s en c e o f m al t o s e. Ad d i t i o n o f m al t o s e t o M BP -A9 6 W / I3 2 9 W b ro u gh t ab o u t a s i gn i fi c an t s t i m u l at i o n o f t h e M al F GK 2 ATP as e, al t h o u gh i t was m u ch l e s s t h an s t i m u l at i o n b y w i l d -t yp e M BP. Th es e m e as u rem en t s wer e m ad e at an M BP co n cen t r at i o n o f 2 µ M, b u t t h e s am e r es u l t s wer e ob t ai n ed at 1 µ M an d 5 µ M M BP co n cen t r at i o n s (n o t s h o wn ). In t h e p r es en c e o f m al t o s e, i n creas es i n t h e co n cen t rat i o n o f al l t h ree p ro t ei n s p ro d u c ed i n cr ea s es i n s t i m u l at i o n o f t h e Mal FGK 2 ATP as e, b u t M BP -DM co n s i s t en t l y ex h i b i t ed a m o d es t l y co m p ro m i s ed ab i l i t y t o s t i m u l at e M al FGK 2 co m p a red t o wi l d -t yp e M BP, wh i l e M BP -A9 6 W / I3 2 9 W was s ev erel y c o m p ro m i s ed. Th u s, m u t at i o n s d es t ab i l i z i n g t h e op en co n fo rm at i o n d o n o t en h an c e t h e ab i l i t y o f t h e p ro t ei n s t o s t i m u l at e t h e M al FG K 2 ATP as e i n t h e ab s en ce o f m al t o s e, an d i n f act red u ce t h e m al t o s e-s t i m u l at ed M al FGK 2 ATP as e act i v i t y. In t h i s regard, we h av e co n s i d ered t h e p o s s i b i l i t y t h at d ecr eas es i n t h e ab i l i t y o f t h e m u t an t s t o s t ab i l i z e t h e t ran s i t i o n s t at e m i gh t h av e o ffs et i n creas ed s t i m u l at i o n b y h i gh er p rop o rt i o n s o f t h e cl o s ed co n fo rm at i o n. I b e l i ev e t h i s i s h i gh l y 68

80 Fig 2.8 Destabilization of the Open Conformation Decreases MalFGK 2 ATPase Th e ab i l i t y o f wi l d -t yp e M BP (M BP -W T), M BP - DM, an d M BP -A9 6 W / I3 2 9 W t o s t i m ul at e t h e M al FGK 2 ATP as e was m eas u red i n ei t h er t h e ab s en ce (h o l l o w b ars ) o r p res en c e (fi l l ed b ars ) o f 5 m M m al t o s e. An M BP co n c en t rat i o n o f 2 µ M was us ed i n each c as e, an d m eas u r em en t s were m ad e i n t ri p l i cat e; t h e m ean v al u e i s s h o wn wit h erro r b a rs rep r es en t i n g t h e s t an d ard d ev i at i o n. Th e P LS o n t h ei r o wn (h o l l o w b ar, fa r l eft ) s h o w a l o w b as al ATP as e t h at i s d o ub l ed b y ad d i t i o n o f u n l i gan d e d M BP an d i n cr eas ed o v er 1 0- fo l d b y ad d i t i o n o f M BP an d m al t o s e. In t h e p res en ce o f m al t o s e, M BP -DM d o es n o t s t i m u l at e t h e M al FGK 2 ATP as e t o t h e s am e d e gr ee as wi l d -t yp e M BP. M BP -A9 6 W / I3 2 9 W s h o ws a s ev erel y i m p ai r ed ab i l i t y t o s t i m u l at e M al FGK 2 i n b o t h t h e ab s en ce an d p res en c e o f m al t o s e. 69

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82 u n l i k el y. Bo t h o f t h e m u t an t s h av e l i gan d b i n d i n g affi n i t i es t h at ar e at l eas t 2 o rd ers o f m a gn i t u d e h i gh er t h an wi l d t yp e, an d o n t h i s b as i s t h e op en -cl o s ed eq u i l i b ri u m h as b een s ev e rel y p e rt u rb ed. As s u m i n g t h e d es t ab i l i z in g m u t at i o n s h av e t h e s a m e eff ect o n t h e u n l i gan d ed p ro t ei n as t h e y d o o n t h e l i gan d -b o u n d p ro t ei n, t h en o n e wo u l d ex p ect a s i gn i fi can t (up t o 2 o rd ers o f m a gn i t u d e) i n creas e i n t h e fra ct i o n o f cl o s ed u n l i g an d ed M B P in s o l u t i o n. In co n t r as t, d es t ab i l i z at i o n o f t h e op en co n fo rm at i o n p ro d u ces o n l y a m o d es t 2 0 % d ecre as e i n m al t o s e-s t i m u l at ed ATP as e a ct i v i t y fo r M BP -DM, an d a 5 -fo l d d e cre as e fo r M B P -A9 6 W / I3 2 9 W. Th er ef o re i t i s u n l i k el y t h at i n creas es i n t h e p rop o rt i o n o f t h e cl o s ed co n fo rm a ti o n o f t h es e m u t an t s - an d as s o ci at ed i n cr eas es i n M al FGK 2 s t i m u l at i o n - ar e b ei n g co m p l et el y m as k ed o r o f fs et b y t h ei r d ecr eas ed ab i l i t y t o s t ab i l i z e t h e t ran s i t i o n s t at e. In s u m m a r y, t h e cl o s ed c o n fo rm at i o n i s m o s t l i k ely n o t res p o n s i b l e fo r s t i m u l at i o n o f t h e ATP as e i n t h e ab s en ce o f m al t o s e. In ad d i t i o n, t h e res u l t s wi t h t h e co n fo rm a t i o n al l y en gi n e ered M BP m u t an t s s h o w t h at t h e en er g et i cs o f d o m ai n op eni n g, an d t h e s t ab i l i t y o f t h e op en co n fo rm at i o n, ar e i m p o rt an t fo r ATP h yd ro l ys i s. 71

83 2.4 Discussion Th e m ech an i s m o f s ub s t rat e i m p o rt b y A BC i m p o rt s yst em s i n v o l v es b i n d i n g o f l i gan d t o t h e p eri p h e ral b i n d i n g p ro t ei n t o ex ert a co n f o rm ati o n al ch an g e t h at s i gn al s t o t h e i n t e g ral m em b ran e s ub u n i t s t o i n i t i at e ATP h yd ro l ys i s. Th e sh ap e co m p l em en t a ri t y o f t h e i n t er act i n g s u rfac es - cl o s ed M BP a n d an u n d efi n ed co n fo rm at i o n o f M al FGK 2 - i s cri t i cal, an d t h i s h as b e en d em o n s t rat ed t h ro u gh m u t agen i c s t u d i es [ 2 5,2 6 ]. Al on g wi t h m an y o t h er p eri p h e ral b i n d i n g p ro t ei n s, u n l i gan d ed M B P ex i s t s i n an op en co n fo rm at i o n i n s o l u ti o n [ 1 5, ], an d t h erefo re i t h as al wa ys b e en d i ffi cu l t t o u n d ers t an d h o w t h e u n l i gan d ed b i n d i n g p ro t e i n i s ab l e t o s t i m u l at e ATP h yd ro l ys i s b y t h e t ran s p o rt er, an ef fect d o cu m en t ed fo r t h e t wo s ys t em s t h at h av e b e en t es t ed i n t h i s regard - t h e m al t o s e an d h i s t i d i n e tran s p o rt ers [ 4,5 ]. On e p o s si b l e ex p l an at i o n i s t h at t h e u n l i gan d ed b i n d i n g p ro t ei n i s ab l e t o m i m i c th e cl o s ed co n fo rm at i o n o f t h e p ro t ei n. In o t h er wo rd s, t h e s ys t em fo l l o w s a l o ck an d k e y m o d e l : as l o n g as t h e b i n d i n g p ro t ei n h as t h e co rre ct s h ap e, i t s h o u l d b e ab l e t o act i v at e t h e M al FG K 2 ATP as e. Ev i d en c e f o r a cl o s ed, u n l i gan d ed co n fo rm at i o n i n cl u d es c r ys t al l i z at i o n o f t h e gl u co se/ gal act o s e b i n d i n g p ro t ei n i n an u n l i gan d ed, cl o s ed co n fo rm at i o n [ 1 9 ], an d, m o re r ecen t l y, t h e C h o X p ro t ei n fro m S. meliloti [ 2 0 ]. In ad d i t i o n, an al t ern at e s o l u t i o n co n fo rm at i o n fo r t h e m al t o s e b i n d i n g p ro t ei n (M BP ) h as b een d o cu m en t ed b y NM R [ 1 8 ] an d i n s o l u t i o n t h e o v eral l co n fo r m at i on o f u n l i g an d ed M BP ap p e ars t o b e s l i gh t l y m o re cl o s ed t h an t h e u n l i gan d ed cr ys t al s t ru ct u r e [ 1 5,2 7 ] h i n t i n g t h at M BP m a y ex i s t, t o s o me d eg ree, i n cl o s ed co n fo r m at i o n (s ) i n s o l u t i o n. Ap art fro m t h es e r es u l t s, wh i ch are co n s i s t en t wi t h t h e ex i s t en ce o f a s m al l fract i o n o f a cl o s ed, u n l igan d ed fo rm o f t h e b i n d i n g p ro t ei n i n s o l u t i o n, t h ere i s n o ex p eri m en t al ev i d en ce fo r t h e s t i m u l at io n o f t h e m em b ran e ATP as e b y a cl o s ed, u n l i gan d ed co n fo rm at i o n o f t h e b i n d i n g p ro t ei n. Th e i d ea i s b as ed s o l e l y o n t h e fact t h at t h e cl o s ed, l i gan d ed fo rm i s r es p o n s i b l e fo r s t i m u l at i on i n t h e p res en c e o f l i gan d. 72

84 Th i s s t u d y was i n i t i at ed t o t es t wh et h er act i v at i o n o f t h e M al FGK 2 ATP as e b y u n l i gan d ed M BP was d u e t o a cl o s ed u n l i gan d ed co n fo rm at i o n. Our h op e was t h at t h i s i n fo rm at i o n wo u l d h el p u s t o u n d ers t an d t h e m o l ecu l ar m ech an i cs o f t he s ys t em, an d, m o r e s p eci fi cal l y, t o ad d res s t h e ro l e o f m al t o s e i t sel f i n t h e t ran s p o rt p ro ces s. Ou r res u l t s ar gu e agai n s t t h e i d ea t h at u n l i gan d ed M BP s t i m u lat es t h e t ran s p o rt e r t h ro u gh a cl o s ed co n fo rm at i o n. Th e d ep en d en ce o f t h e s t i m u l at i o n o n M BP co n cen t r at i o n d o es n o t fo l l ow wh at wo u l d b e exp ect ed i f t h i s w ere t h e m ech an i s m. S p eci fi cal l y, t h e u n l i gan d ed fo rm o f t h e p ro t ei n s t i m u l at es M al FGK 2 wi t h a m u ch l o wer K M v al u e t h an m al t o s e-b o u n d M BP, i n d i cat i n g t h at t h e s p e c i es res p o n s i b l e i s n o t m i m i ck i n g t h e cl o s ed co n fo rm at i o n o f M B P. Fu rt h erm or e, d es t ab i l i z at i o n o f t h e op en co n fo rm at i o n - an d co n s eq u en t i n creas e i n t h e prop ort i o n o f t h e cl o s ed co n fo rm at i o n - h ad n o p o s i t i v e effect o n t h e ab i l i t y o f u n l i g an d ed M BP t o s t i m u l at e t h e M al FGK 2 ATP as e. W e co n cl u d e t h at a cl o s ed, u n l i g an d ed co n fo rm a t i o n o f M BP i s n o t res p o n s i b l e fo r M BP - d ep en d en t M al F GK 2 s t im u l at i o n i n t h e ab s en ce o f m al t o s e. On e ex p l an at i o n fo r wh y t h i s i s t h e cas e i s t h at l i g an d i s req u i red t o s t ab i l i z e t h e clo s ed co n fo rm at i o n o f t h e b i n d i n g p ro t ei n. M u ch o f t h e ex p eri m en t al wo rk on t h e m al t o s e t ran s p o rt er i s co n s i s t en t wit h t h e ex i s t en ce o f en er get i c b arri ers t o co n fo rm at i o n al ch an g es i n M al FGK 2 [ 4 1]. Th es e wo u l d rep res en t h i gh e r en er g y i n t erm ed i at es o f M al FG K 2 t h at p rev en t fu t i l e c yc l i n g an d A TP h yd ro l ys i s i n t h e ab s en c e o f m al t o s e. M BP m a y fu n c t i o n t o s t ab i l iz e s u ch i n t erm ed i at es, t h e reb y p ro m o t i n g co n fo rm at i o n al ch an ges i n t h e s ys t em. Ho wev e r, t o s t ab i l i z e a h i gh-en er g y M al FGK 2 i n t erm ed i at e, M BP wo u l d n o t o n l y h av e t o b e i n t h e co rre ct co n fo rm at i o n, b ut i t wo u l d al s o h av e t o b e i n a l o w en e r g y s t at e; t h at i s, a h i gh en e rg y fo rm o f M BP wo u l d n o t co n t ri b u t e t o t h e s t ab i l i z at i o n o f a h i gh en er g y fo rm o f M al FG K 2. W h il e t h e cl o s ed, l igan d ed co n fo rm at i o n re p res en ts a s t ab l e, l o w en er g y fo rm o f M BP, a cl o s ed u n l i g an d ed co n fo rm at i on i s an u n s t ab l e h i gh en er g y fo rm [ 2 3 ], 73

85 an d o n t h i s b as i s, o nl y l i gan d -b o u n d M BP wo u l d b e a go o d can d i d at e fo r s t ab i l i z i n g a h i gh en er g y fo rm o f M al FGK 2. Th e act i v at i o n o f t h e M al FGK 2 ATP as e b y u n l i gan d ed M BP i s m o s t l i kel y d u e t o a hi gh affi n i t y i n t e ract i o n b et w een t h e op en co n fo rm at i o n o f M BP an d a s p ars e l y p op u l at ed M al FGK 2 co n fo rm at i o n. Th e m ax i m u m rat e o f ATP h yd ro l ys i s i n t h e ab s en ce o f m al t o s e wo u l d b e l i m i t ed b y t h e p op u l at i o n o f M al FGK 2 t h at i s i n t he req u i red co n fo rm at i o n. M al FGK 2 h as b een cr ys t al l i z ed i n t wo co n f o rm at i o n al s t at es [ 1 1,1 2 ], an d i n t h e m em b r an e t h ere m a y b e o t h er i n t erm ed i at es. At eq u i l i b ri u m t h e p rop o rt i o n o f t h es e d i ff eren t co n fo rm at i o n s wi l l b e d et e rm i n ed b y t h ei r rel at i v e en er gi e s, an d t h ere m a y al s o b e ki n et i c b arri e rs t o i n terco n v e rs i o n b et ween co n fo rm at i o n s. In ei t h e r cas e, gi v en fi n i t e l i m i t s on t h e en er gi es o f t h e co n fo rm at i o n s an d k i n et i c b arri ers, a s m al l p rop o rt i o n o f t h e M al FG K 2 p op u l at i o n wi l l fi n d i t s el f i n h i gh e r en er g y s t at es, an d t h es e a re t h e m o l e c u l es t o wh i ch op en M B P co u l d b i n d, p ro m o t i n g t h e ATP as e a ct i v i t y b y s t ab i l i z i n g t h e t ran s i t i o n s t at e o r s o m et h i n g cl o s e t o i t. Th e op en, u n l i g an d ed co n fo rm at i o n i s t h e s eco n d l o w en er g y fo r m o f M BP (aft er t h e cl o s ed, l i gan d ed co n fo rm at i o n ), an d t h er efo r e wo u l d b e ab l e t o s t ab i l i z e a h i gh er en er g y co n fo rm at i o n of M al FGK 2. W e h av e d em o n s t rat ed t h at d es t ab i l i z at i o n o f t h e op en M BP co n fo rm at i o n res u l ts i n a d ecreas ed ab i l i t y o f M BP t o s t i m u l ate t h e M al FGK 2 ATP as e, co n s i s t en t wi t h t h e i d ea t hat t h e op en co nfo rm at i o n o f M BP co n t ri b u t es t o t h e s t ab i l i t y o f t h e t ran s i t i o n s t at e co m p l ex [ 1 1]. In ad d i t i o n t o b ei n g co n s i s t en t wi t h t h e ob s erv at i on s i n t h i s s t u d y, t h e i d ea o f d i rect k i n et i c s t ab i l i z at i o n o f a h i gh en er g y fo rm o f M al FGK 2 b y op en M BP ex p l ai n s an ob s erv at i o n m ad e b y M eri n o an d S h u m an [ 4 2 ]. In t h i s s t u d y, a M al F G K 2 m u t an t was i s o l at ed t h at was ab l e t o t ran s p o rt l act o s e, b u t o n l y i n t h e p res en c e o f M BP, wh i ch d o es n o t b i n d l act o s e. Th i s wo rk co n fi rm ed t h e i m p o rt an ce o f t h e i n t e ract i o n b et we en u n l i g a n d ed M BP an d t h e m em b ran e co m p o n en t s. Fu rt h erm o re, gi v en i t s ro b u s t s t i m u l at i o n o f l act o se t ran s p o rt an d t h e f act t h at t h e v as t m aj o ri t y o f 74

86 u n l i gan d ed M BP i s i n an op en co n fo rm at i o n, i t i s alm o s t cert ai n l y t h e op en co n fo rm at i o n o f M BP t h at i s req u i red fo r l act o s e t ran s p o rt i n t h i s s ys t em. 75

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90 Chapter 3 Studies of the Maltose Transport System Reveal a Mechanism for Coupling ATP Hydrolysis to Substrate Translocation Without Direct Recognition of Substrate. 3.1 Introduction ATP Bi n d i n g C as s et t e ( ABC ) t ran s p o rt ers m o v e v ari ou s s ub s t rat es a cro s s m em b ran es, wi t h s ub s t rat e m o v em en t co up l ed t o t h e h yd ro l ys i s of ATP. W h i l e t h e ATP as e act i v i t y o f ABC ex p o rt ers l i k e P -gl ycop ro t ei n i s gen e ral l y s t i m u l ated b y s ub s t r at e b i n d i n g, t h e ATP as e a ct i v i t y o f ABC i m p o rt ers i s act i v at ed b y a p eri p h eral s ub s t r ate b i n d i n g p ro t ei n an d n o t t h e free s ub s t r at e [ fo r recen t r ev i ews s e e 1-3 ]. Ho wev er, t h e m e ch a ni s m o f ATP as e re gu l at i o n i s s t i l l n o t fu ll y u n d ers t o o d. Her e, we u s e o n e o f t h e m o s t wel l -s t u di ed ABC i m p o rt ers, t h e Escherichia coli m al t o s e t ran s p o rt er (M al FGK 2 ), t o i n v es t i g at e t h e ro l es o f m al t o s e b i n d i n g p ro tei n (M BP ) an d m al t o s e i t s el f i n re gu l at i o n o f ATP as e a ct i v i t y In i t s res t i n g s t at e M al FG K 2 co n t ai n s a s ub s t rat e-b i n d i n g s i t e t h at i s ex p o s ed t o t h e c yt op l as m [ 4 ]. In t h e p er i p l as m M BP b i n d s m al t os e, wh i ch s t ab i l i z es a ch a n ge fro m an op en t o cl o s ed co n fo rm at i o n, e n ab l i n g i t t o s t i m u l at e t h e M al FGK 2 ATP as e [ 5,6 ]. In t e ra ct i o n s wi t h cl o s ed, m al t o s e-b o u n d M BP l ead t o ex p o s u re o f t h e M al FG K 2 m al t o s e-b i n d i n g s i t e t o t h e p eri p l as m i c s i d e wh e re m al t o s e can m o v e f ro m M BP i nt o an o ccl u d ed t r an s l o cat i o n p at h wa y [ 7,8 ]. Aft er ATP h yd ro l ys i s t h e t ran s p o rt er r et u r ns i t s b i n d i n g s i t e t o t h e cyt op l as m i c f ace t o al l o w t h e s ub s t rat e t o en t er t h e c yt op l as m. Th i s is k n o wn as t h e al t ern at i n g acc es s m o d el o f m al t o s e t ran s p o rt [ 4 ] an d m a y b e a co m m o n m e ch an i s m am o n g A BC t ran s p o rt ers [ 2,9,1 0 ]. Th e s t ru ct u re o f a t r an s i t i o n s t at e co m p l ex b et wee n M BP an d M al F GK 2, as wel l as b i o ch em i cal d at a [ 7,8 ] i n d i cat es t h at m al t o s e en t e r s t h e s ub s t rat e b i n d i n g s i t e o f M al F GK 2 p ri o r t o ATP h yd ro l ys i s, b u t i t i s u n cl ear h o w m al t o s e -b ou n d M BP act i v at es t h e M al FGK 2 ATP as e 79

91 [ 1 1 ] an d h o w ATP h yd r o l ys i s i s co up l ed t o t h e m o v em en t o f m al t o s e a cro s s t h e m em b ran e. O f p art i cu l ar i n t er es t ar e t h e ro l es t h at m al t o s e i t s el f m i gh t p l a y i n re gu l at i n g t h e ATP as e a ct i v i t y o f M al FGK 2. Th ere are t wo w a ys m al t o s e co u l d re gu l at e ATP h yd ro l ys i s : t h e fi rs t i s b y s t ab i l i z i n g t h e cl o s ed co n fo rm at i o n o f M BP an d t h e s e co n d i s t h ro u gh d i r ect i n t er act i o n s wi t h M al FGK 2. W h i l e i t i s cl ear fro m p r ev i o u s s t u d i es t h at s ub s t rat e i nd u ced d o m ai n cl o s u r e i n M BP i s cri t i cal fo r rob u s t s t i m u l at i o n o f M alfgk 2 ATP as e a ct i v i t y an d s ub s t rat e t ran s p o rt [ 6 ], i t i s n o t k n o wn wh et h er d i r ect i n t era ct i o n b et ween m al t o s e an d M al FGK 2 i s al s o req u i re d fo r ATP as e act i v i t y. To ad d res s t h i s q u es t i o n, we h av e u s ed an M BP m u t an t t h at i s ab l e t o b i n d an al t ern at e s ub s t rat e, s u cro s e, wi t h h i gh af fi n i t y [ 1 2 ]. Th e su cro s e-b i n d i n g M BP (s M BP ) en ab l es u s t o p res en t t h e m al t o s e t r an s p o rt er wi t h ei t h er m al t o s e o r s u cro s e i n eq u i v al en t co n t ex t s an d d i s t i n gu i s h wh et h er t h e s ub s t i t u t i o n i n fl u en ces t h e ATP as e act i v i t y o f M al F GK 2. S u cro s e i s a go o d al t e rn at e s ub s t r at e f o r t h i s p u rp o s e b ec au s e e x p eri m en t s fro m S h u m an s l ab o rat o r y h av e s h o wn t h at i t h as a v er y p o o r ab i l i t y t o co m p et e for t h e m al t o s e b i n d i n g s i t e i n M al FGK 2, i n d i cat i n g t h at t h e ch an g e i n s u ga r s t ru ct u r e i s s uf fi ci en t t o d i s rup t s p eci fi c b i n d i n g i n t era ct i o n s wi t h M al FGK 2 [ 1 3,1 4 ]. Us i n g s M BP w e h av e d et erm i n ed t h at ATP h yd ro l ys i s b y M al F GK 2 i s n o t d ep en d en t o n t h e ex act n at u re o f t h e s u b s t rat e, an d t h er efo r e t he co up l i n g o f ATP as e a ct i v i t y t o s ub s t rat e t ran s l o cat i o n i s d u e s o l el y t o i n t era ct i o n s b et we en M BP an d M al F GK 2. Ba s ed o n t h es e fi n d i n gs an d d et ai l ed s t ru ct u r al an al ys i s o f s M BP, we p rop o se t h at a p ro d u ct i v e i n t e ract i o n b et we en M al G an d t h e v ac at ed m al t o s e b i n d i n g s i t e i n M BP i s re q ui red fo r ATP h yd ro l ys i s. In t h i s m an n e r, s ub s t rat e t ran s l o cat i o n f r o m M BP t o M al FGK 2 i s co up l ed t o ATP h yd ro l ys i s wi t h o u t req u i ri n g a d i rect i n t era ct i o n b et wee n m al t o s e an d M al FG K 2. 80

92 3.2 Materials and Methods Cloning of MBP mutants P l as m i d p D IM -C 8 M al E, co n t ai n i n g s M BP, was k i n d l y p ro v i d ed b y t h e Os t erm ei er g ro up [ 1 2 ]. A b p Kp n 2 1 / Bcl 1 fra gm en t o f t h i s v e ct o r, co n t ai n i n g t h e W 6 2 Y an d E1 1 1 Y s ub s t i t u t i o n s, was l i gat ed i n t o p LH1, wh i ch co n t ai ns t h e M BP s i gn al s eq u e n ce fo r ex p o rt t o t h e p eri p l as m. Th e D1 4 L an d K1 5 F m u t at i o n s wer e s ub s t i t u t ed b y m u t a g en i c P C R u s i n g p ri m ers : 5 -C TGGATTA AC GGC C TTTTC GGC TATAAC GGTC TC GC -3 an d 5 -GC GAGAC C GTTA T AGC C AAAAAGGC C G TTAATC C AG-3. To p ro d u ce i n t ra cel l u l arl y ex p res s ed s M BP an d w t M BP wi t h a h ex a-h i s t i d in e affi n i t y t a g, res t ri ct i o n cu t s i t es fo r E h e1 an d Hi n d 3 we re ad d e d t o ex ci s e t h e t wo gen es (wi t h o u t l o cal i z at i o n t ag) u s i n g p ri m ers : 5 C GC C TC GGC TGGC GC C AAAATC GAAG -3 an d 5 C GC C GC ATC C GGCATTTAAGC TTATT AC TTGGTGATAC G AG-3 Di ges t ed P C R p ro d u ct s were t h en l i gat ed i n t o t h e m ul t i cl o n i n g s i t e o f p P R OEX-HT a ( In v i t ro gen ) t o i n t ro d u ce an N-t erm i n al h ex a-h i s t i d i n e t ag at t a ch ed b y a t ob a cco et ch v i ru s (TEV) p ro t eas e cl eav ab l e l i n k er. C l eav a ge o f t h i s l i n k er l eft an N -t erm i n al gl yc i n e- al an i n e i n s ert i o n t h at was co m m o n t o b o t h t h e s M BP an d wt M BP u s ed i n t h i s s t u d y. Growth on Minimal Media Bact eri al s t rai n HS , l ack i n g en d o gen o u s wt M BP p ro d u ct i o n, was t r an s fo rm ed wi t h n o p l as m i d, p LH1 co n t ai n i n g wt M BP, o r p LH 1 -S co n t ai n i n g s M BP. Th es e s t rai n s were s t r eak ed o n M 9 m i n i m al m ed i a au gm en t ed wi t h 5 % m al t o s e an d in cub at ed at 3 7 C f o r up t o 7 d a ys. C o l o n y fo rm at i o n was ob s erv ed an d reco rd ed. 81

93 Expression and Purification Hex a-h i s t i d i n e t ag ged s M BP an d wt M BP w ere e x p res s ed an d p u ri fi ed f ro m HS (M al E -/ - ) E. coli b y Ni 2 + -affi n i t y ch ro m at o gr ap h y. R em o v al o f t h e af fi n i t y t ag w as ac co m p l i s h ed b y TEV p ro t eas e cl e av a g e, an d p u re s M BP an d wt M BP was ob t ai n ed b y i o n ex ch an g e ch ro m at o grap h y, as p rev i o u s l y rep o rt ed [ 1 5 ]. Bo t h p ro t ei n s wer e d en at u r e d i n 6 M gu an i d i n e an d d i al yz ed ex h au s t i v el y t o rem o v e t ra ce s u ga rs b efo re b ei n g re fo l d ed b y d rop wi s e d i l u t i o n i n 5 0 m M Tri s -HC l p H 8, co n cen t rat ed, an d s t o red i n t he s am e b u ff er [ 1 5 ]. Preparation of wt-malfgk 2 -containing proteoliposomes M al FGK 2 was o v e r-ex p r es s ed fro m p l as m i d s p N T1 S K+ an d p M R i n E. coli HS cel l s, wh i ch co n t ai n d el et i o n s fo r al l t ran s p o rt er co m p o n en t s. M em b ran e f r act i o n s wer e p rep ared an d s o l ub i l i z ed as p rev i o u s l y rep o rt ed [ 1 5 ]. Li p o s o m es w ere p rep a re d fro m Av an t i cru d e E. coli p h o s p h o l i p i d s an d aft er h o m o gen i z at i o n b y s o n i c at i o n were co m b i n ed wi t h M alfgk 2 -co n t ai n i n g m em b ran es b y d et er gen t d i l u t i o n [ 1 5 ]. Th e p ro t eo l i p o s o m es wer e fro z en at -8 0 C u n d er N 2 u n t i l u s ed. ATPase Assays ATP as e m eas u r em en t s w ere m ad e i n a s o l u t i o n o f 5 0 m M Tri s -HC l p H 8.0, m M KC l, an d 1 0 m M M gc l 2, wi t h p ro t eo l i p o s o m es ad d ed t o a fi n al co n c en t rat io n o f 0.1 m g/ m L p ro t ei n. P u ri fi ed s M BP o r wt M B P was ad d ed at v ari o u s c o n cen t rat i o n s an d i n t h e p res en c e o r ab s en c e o f 5 m M m al t o s e o r s u cro s e. ATP h yd ro l ys i s at 3 7 C was m eas u red in vitro b y as s a yi n g t h e ap p ear an c e o f i n o r gan i c p h o s p h at e, u s i n g am m o n i u m m o l yb d at e, as p r ev i o u s l y d es c ri b ed [ 1 5 ]. 82

94 Crystallization and X-ray Diffraction C r ys t al s o f s u c ro s e-b o u n d s M BP were g ro wn i n 2 1 % P EG K, m M s o d i u m acet at e p H 6.5, 6 0 m M M gc l 2 an d 1 0 m M Zn C l 2 wi t h m M s u cro s e. C r ys t al s o f u n l i gan d ed s M BP were g ro wn f ro m P EG 5 k -M M E, s o d i u m acet at e p H 6.5, 6 0 m M M gc l 2 an d 1 0 m M Zn C l 2. C r ys t al s o f s u c ro s e-b o u n d an d u n l i gan d ed s M BP d i ffra ct ed t o 2 Å an d 1.5 Å, res p ect i v el y, at t h e C an a d i an Li gh t S o u rc e b eam l i n e C M C F1 (0 8 ID-1 ). P h as es wer e d et erm i n ed b y m o l ecu l ar r ep l acem en t wit h t h e wi l d -t yp e p ro t e i n s (P DB- ID 1 ANF an d 1 OM P ). R i gi d b o d y r efi n em en t o f t h e t wo i s o l at ed d o m ai n s wa s carri ed o u t fi rs t t o cap t u re an y d o m ai n m o v em en t s rel at i v e t o t h e wi l d -t yp e s t r u ct u res. S t ru ct u res we re r efi n ed u s i n g C NS [ 2 1] (Tab l e 1 ) wi t h m an u al adj u s t m en t i n C OOT [ 2 2 ]. Th e s t ru ct u res h av e b een d ep o s i t ed i n t h e P DB wi t h IDs 3 KJ T an d 3 HP I fo r t h e op en an d cl o s ed fo rm s res p e ct i v el y. M o l e cu l ar fi gu r es we re m ad e u s i n g P ym O L [ 2 3 ]. Small Angle X-ray Scattering S AXS ex p eri m en t s were carri ed o u t at Bi o C AT b e am l i ne ID1 8 at t h e Ad v a n ced P h o t o n S o u rce (A r go n n e Il l i n o i s, U.S.A.) as p rev i o u s l y d e s cri b ed [ 2 5 ]. 83

95 3.3 Results A sucrose-binding MBP S u cro s e-b i n d i n g M al t o s e Bi n d i n g P ro t ei n (s M BP ) i s a m u t an t fo rm o f M B P d ev el op ed b y M arc Os t e rm ei er s Gro u p [ 1 2 ]. Th e s M BP m o l ecu l e h as fo u r p o i n t m u t at i o n s, D1 4 L, K1 5 F, W 6 2 Y an d E1 1 1 Y, al l wi t h i n t h e s ub s t rat e b i n d i n g cleft. W h i l e wi l d -t yp e M BP (wt M BP ) h as a d i s s o ci at i o n co n s t an t (K D ) o f 1 µ M fo r m al t o s e an d n o m eas u rab l e ab i l i t y t o b i n d s u cro s e [ 6 ], s M BP h as a K D fo r m al t o s e o f 2 4 µ M an d fo r s u c r o s e o f 6.6 µ M [ 1 2 ]. Th ese v al u es we re co n fi rm ed fo r m y s M BP co n s t ru ct s u s i n g fl u o res c en ce t i t rat i o n s. Fu rt h erm o re, we m e as u red s ub s t rat e i n d u ced co n fo r m at i o n al ch an ges i n s M B P, i n s o l u t i o n, b y s m al l a n gl e X -ra y s cat t eri n g. S u cro s e-i n d u c ed ch an g es i n t h e co n fo rm at i o n o f s M BP wer e i d en t i cal t o ch an g es s een i n wt M BP (Fi gu re 3.1 ). I al s o ob s e r v ed t h at s M BP co u l d p art i al l y co m p l em en t t h e g ro wt h o f wt M BP d efi ci en t E. coli o n M 9 m al t o s e m i n i m al m ed i a, res u l t i n g i n co l o n y fo rm at i o n o n ag ar p l at es aft er 2 d a ys, rat h er t h an o v ern i gh t gro wt h as s een wi t h wt M BP, o r n o gro wt h, as s een i n t h e ab s en ce o f b i n d i n g p ro t ei n ex p res s i o n. Al t h o u gh gro wt h w as s l o w co m p ared t o wi l d -t yp e, i t w as cl ea r t h at s M BP co u l d i n t eract p ro d u ct i v el y wi t h M al FGK 2 t o p ro m o t e m al t o s e t ran s p o rt. smbp stimulates MalFGK 2 with bound sucrose or maltose M al FGK 2 h as a b i n d i n g s i t e t h at i s rel at i v el y s p eci fi c fo r m al t o d ex t ri n s [ 1 3 ], an d b i n d i n g o f t h e s ub s t rat e t o t h i s s i t e m a y b e i m p o rt an t fo r s t i m u l at i o n o f t h e M al FG K 2 ATP as e. To d et erm i n e t h e i m p o rt an c e o f s p eci fi c i n t e ract i o n s b et ween m al t o s e an d M al FGK 2, we u s ed s M BP t o p res en t M al F GK 2 wi t h ei t h er m al t o s e o r s u c ro s e as a t ran s p o rt s ub st r at e, an d m eas u r ed t h e res u l t i n g s t i m u l at i o n o f ATP as e act i v i t y in vitro. C o n s i s t en t wi t h l it erat u r e fi n d i n gs, M al FGK 2 - co n t ai n i n g p ro t eo l i p o s o m es s h o wed a l o w l ev el o f b as al a ct i v i t y ( Fi gu re 3.2 ). Th i s act i v i t y w as u n al t ered b y t h e ad d i t i o n o f 5 m M m al t o s e o r s u cr o se, co n fi rm i n g t h at fr ee s u ga r can n o t s t i m u l at e 84

96 Fig 3.1 Open to Closed Conformational Change in smbp S m al l an gl e X- ra y s c at t er i n g (S AXS ) f ro m p u ri fi e d s M BP was m eas u r ed i n t h e ab s en c e (d as h ed cu rv e) an d p res e n ce (s o l i d cu rv e) o f 5 m M su cro s e. Th e i n s et s h o ws t h e d i ffer en c e b et ween t h e t wo cu rv es, ex p res s ed as a p e rcen t a g e of t h e t o t al s i gn al. S u cr o s e b i n d i n g b ri n gs ab o u t a d ec reas e i n t h e ra d i u s o f g yr at i o n fro m t o Å; t h e cu rv es fo r s M BP i n t h e ab s en ce an d p r es en c e o f s u cro s e m at ch t h o s e o f w t M BP i n t h e ab s en ce an d p res en c e o f m al t o s e. 85

97 86

98 t h e t ran s p o rt er i n t h e ab s en ce o f M BP [ 1 1 ]. W h en 20 µ M s M BP was ad d e d t o wi l d t yp e M al FGK 2, n o s t at i s t i cal l y s i gn i fi can t i n c reas e i n a ct i v i t y was ob s e rv ed. Ho wev er, i n t h e p res en ce o f either 5 m M m al t o s e o r s u cro s e, s M BP s t i m u l at ed a 4 -fo l d i n c reas e i n A TP h yd ro l ys i s o v er b ack gro u n d ( Fi gu r e 3.2 A, d i ago n al fi l l ). W e h av e t h erefo r e ob s erv ed t h at, wi t h res p ect t o ATP as e act i v at i o n, M al F GK 2 can n o t d i s t i n gu i s h s u cro s e f ro m m al t o s e. To d em o ns t rat e t h at t h e eq u i v al en c e o f m al t o s e- an d s u cro s e -b o u n d s M B P was n o t l i m i t ed t o 2 0 µm s M BP co n cen t r at i o n, a ran ge o f co n cen t rat i o n s, fro m 1 t o µ M, were t es t ed (Fi gu r e 3.2 B ). Ac ro s s t h i s co n cen t r at i o n ran ge, m al t o s e- an d s u c ro s e-b o u n d s M BP s t i m u l at e t h e M al F GK 2 ATP as e t o s i m i l ar l ev el s ; t h e o v eral l t ren d - i n b o t h cas es a p r op o rt i o n al i n creas e i n M al FGK 2 ATP as e act i v i t y as s M BP co n c en t rat i o n i s rai s ed - s u g ges t s t h at t h e m ech an i s m fo r s t i m u l at i o n i s t h e s am e, i rres p ect i v e o f wh i ch s u g ar M al FGK 2 co m e s i n co n t act wi t h. Al t h o u gh m al t o s e- an d s u cro s e-b o u n d s M BP b o t h s t i m u l at e t h e M al FGK 2 ATP as e t o t h e s am e ex t en t, t h e ab s o l u t e l ev el s o f ATP as e act i v i t y p ro d u ced b y s M BP wer e m u ch l o wer t h an t h o s e p ro d u ced b y wt M B P. Fo r ex am p l e, i n t h e p r es ence o f m al t o s e t h e l ev el o f ATP as e s t i m u l at i o n b y 2 0 µ M wt M BP was 4 0 fo l d h i gh er t h an t h e s t i m u l at i o n p ro d u ced b y t h e s am e co n cen t r at i o n o f ei t h er m al t o s e- o r s u cr o s e-b o u n d sm BP. In ad d i t i o n, u n l i g an d ed s M BP d i d n o t p ro d u ce a s i gn i fi c an t i n cr eas e i n M al FGK 2 ATP as e, i n co n t ras t t o u n l i g an d ed wt M BP wh i ch co n s i s t en t l y p ro d u c es a 2 -fo l d s t i m u l at i o n (Fi gu r e 3.2 A)[ 1 1,1 5 ]. Th erefo re, wh i l e t h e s ub s t i t u t i o n o f s u cro s e fo r m al t o s e d i d n o t i n fl u ence s t i m u l at i o n o f t h e M al FGK 2 ATP as e, wh en co m p ared t o wt M BP t h e m u t at i o n s i n s M BP h av e d ras t i cal l y co m p ro m i s ed i t s o v eral l ab i l i t y t o s t i m u l at e t h e M al FGK 2 ATP as e. Structural analysis of open and closed smbp To d et erm i n e h o w t h e m u t at i o n s i n s M BP d i s rup t i t s ab i l i t y t o s t i m u l at e M al FGK 2, I 87

99 Fig 3.2 Effect of Ligand on Stimulation of the MalFGK 2 ATPase M al FGK 2 was r eco n s t i t u t ed i n t o a p ro t eo l i p o s o m e s ys t em (P LS ) an d t h e eff ect o f M BP an d l i gan d s o n t h e M al F GK 2 ATP as e wer e m eas u red. (A ) Th e P LS h av e a b as al ATP as e (cl e ar b ar) t h at i s n o t s i gn i fi c an t l y i n cre as ed b y u n l i gand ed s M BP. W h en b o u n d t o ei t h er s u cro s e o r m al t o s e, s M BP p ro d u ces a 4 -fo l d s t i m u l at i o n o v er t he b as al rat e (b a rs wi t h d i ago n al fi l l ). Th e m u ch l ar ger ATP as e s t i m u l at i o n b y 2 0 µ M wt M B P (b ars wi t h h o ri z o n t al fi l l ) s h o ws t h at t h e m u t at i o n s i n s M BP h av e i m p ai red i t s ab i l i t y t o s t i m u l at e t h e M al FGK 2 AT P as e co m p ar ed t o wt M BP. (B ) Th e ab i l i t i es o f s u cro s e-s M BP (b l a ck ) an d m al t o s e-s M BP ( gr e y) t o s t i m u l at e M al FGK 2 ab o v e i t s b as a l ATP as e wer e co m p a red o v er a r an ge o f s M BP co n cen t rat i o n s ; t h e s u ga rs wer e p r es en t at a co n cen t r at i o n o f 5 m M. 88

100 89

101 s o l v ed t h e cr ys t al s t ru ct u res o f s M BP i n b o t h t h e s u cro s e-b o u n d an d s ub s t ra t e-fre e fo rm s t o res o l u t i o n s o f 2.0 Å an d 1.5 Å res p e ct i v el y (Tab l e 3.1 ). In b o t h fo rm s s M B P ad op t s a wi l d t yp e fo l d, wi t h m ai n ch ai n at o m s d i fferi n g fro m wt M B P b y an R M S D fo r C A p o s i t i o n s o f Å i n t h e cl o s ed fo rm an d Å i n t h e op en fo rm. W el l -d efi n ed el ect ro n d e n s i t y fo r s u c ro s e w as s ee n i n t h e b i n d i n g s i t e o f t h e s M BP - s u cro s e s t ru ct u r e; t h e el e ct ro n d en s i t y cl ea rl y d efi n es each h yd ro x yl gro up o f s u cro s e an d d o es n o t fi t m al t o s e (Fi gu re 3. 3 A). Li k e m al t o s e-b o u n d wt M BP, s M BP b i n d s s u cro s e t h ro u gh h yd ro g en b o n d s wi t h ea c h o f t h e t wo s u g ar ri n gs. Th e fi rs t, n o n -r ed u ci n g, gl u co s e u n i t i s co m m o n t o b o t h m al t o s e an d s u cro s e an d o c cup i es an i d en t i cal b i n d i n g p o c k et i n wt M BP an d s M BP (Fi gu r e 3.3 B ). Th e s eco n d s u ga r ri n g d i ff er s b et ween m al t o s e an d s u cro s e, b ei n g an α-1,4 l i n k ed red u ci n g gl u co s e i n m al t o s e an d an α -1,2 l i nk ed fru ct o s e i n s u cro s e. As a res u l t, s u cro s e ad op t s a 9 0 b en d co m p a red t o m al t o s e. Th i s b en d al l o ws t h e C 3 h yd ro x yl t o h yd ro g en b o n d wi t h res i d u e W 6 2 Y, wh i ch was l i k el y s el e ct ed fo r t hi s p u rp o s e (Fi gu r e 3.3 B). Th e b en d al s o creat es a c av i t y i n t h e b i n d i n g s i t e an d s ep arat es t h e s u g ar f ro m res i d u es 1 4 an d 1 5 (Fi gu r e 3.3 C ). Th e D1 4 L, K1 5 F an d E1 1 1 Y m u t at i o n s m o d i f y t h i s h yd rop h ob i c cav i t y b y rem o v i n g wh at wo u l d o t h erwi s e b e u n m at ch ed b u ri ed ch a r ges o r p o l a r g ro up s. W e al s o s o l v ed t h e cr ys t a l s t ru ct u re o f t h e op en co n fo rm at i o n o f s M BP t o 1.5 Å res o l u t i o n, an d wer e s u rp ri s ed t o fi n d a s i gn i fi cant ch an ge i n t h e s t ru ct u re o f t h e l i gan d b i n d i n g s i t e. Th e W 6 2 Y m u t at i o n i s ab l e t o ad op t an al t er nat e co n fo rm at i o n t h at d i s p l aces t h e m ai n ch ai n s egm en t fro m res i d u es Y 6 2 t o G6 9 (Fi gu re 3.4 ). Th i s d i fferen ce w as ev i d en t fro m cl e ar el e ct ro n d en s i t y fo r t h e Y6 2 s i d e ch ai n i n t wo d i ffer en t p l a ces, o n e o f wh i ch n e ces s ari l y d i s p l a ces F6 7 an d i s t h erefo r e i n co m p at i b l e wi t h t h e wi l d -t yp e m ai n ch ai n co n fo rm at i o n (Fi gu re 3.4 C ). Th e o n l y wa y t h i s ch an ge can b e a cco m m o d at ed i s fo r res i d u es t o p art i al l y ex t en d i n t o t h e s ub s t rat e b i n d i n g cl eft. Th e o c cup a n ci es o f t h e t wo co n fo rm ati o n s o f res i d u es 6 6 t o 6 9 were s et s u ch t h at 90

102 Table 3.1 Crystallographic Statistics s M BP -S u cro s e Un l i gan d ed s M BP P DB- ID 3 HP I 3 KJ T R es o l u t i o n l i m it (Å) S p ace Gro up P P 2 1 Un i t cel l P aram et e rs a,b,c (Å ) , , , , α,β,γ (d e g) 9 0, 9 0, , , 9 0 M o l ecu l es p er AU 2 1 S o l v en t co n t en t Un i q u e refl ect i o n s a M ean I/ (σ I) (2.3 8 ) (2.0 3 ) a R m erge ( ) ( ) a C o m p l et en es s (%) (8 6.4 ) (5 8.5 ) a R ed u n d an c y 5.8 (4.4 ) 3.3 (2.2 ) Nu m b er o f r efl e ct i o n s To t al R free R efi n em en t R es o l u t i o n ran ge (Å) R wo rk R free R M S D b o n d l en gt h (Å) R M S D b o n d an gl e (d e g) B- Fact o rs (Å 2 ) P ro t ei n Li gan d N/ A S o l v en t R am ach an d r an An al ys i s M o s t fav o red 9 2 % 9 1 % Al l o wed 8 % 9 % Gen ero u s l y Al l o wed 0 0 Di s al l o wed 0 0 a Val u es i n p aren t h es es re fer t o t h e h i gh es t r es o l u t io n s h el l. 91

103 Fig 3.3 Sucrose Binding by smbp. Th e s t ru ct u re o f s u cro s e b o u n d s M BP was s o l v ed an d refi n ed t o 2 Å res o l u t i o n (Tab l e 1 ); co o rd i n at es fo r wt M BP a n d m al t o s e were fro m P DB- ID 1 AN F [ 2 4 ]. (A) Bo u n d s u cro s e (d a rk gr een ) an d t h e m u t at ed r e s i d u es (p al e gr een ) are s h o w, al o n g wi t h 2 F O - F C el ect ro n d en s i t y fo r t h e s u cro s e (b l u e m es h ) co n t o u red at 2 σ. Th e el ect ro n den s i t y m ap w as cal cu l a t ed u s i n g p h as es fro m t h e p art i al l y refi n ed s t ru c t u re, p ri o r t o t h e ad d i ti o n o f s u cro s e t o t h e b i n d i n g s i t e. (B) H yd ro gen b o n d i n g i n t era ct i o n s b et ween s u c ro s e an d s M BP (t op ) are co m p ar ed t o t h o s e b et ween m al t o s e an d wt M BP (b o t t o m ). H yd ro g en b o n d i n g i n t era ct i o n s t o t h e fi rs t gl u co s e ri n g a re t h e s am e i n b o t h p ro t ei n s. (C ) C o m p ari s o n o f t h e l i g an d b i n d i ng s i t es o f s M BP an d wt M BP. Th e m o l ecu l a r s u rfac e t h at s M BP an d w t M BP h av e i n co m m o n i s s h o wn i n gr e y; ca rb o n at o m s fro m m al t o s e an d wt M BP ar e s h o wn i n o ran ge an d ye l l o w, res p ect i vel y, wh i l e t h o s e fro m s u cro s e an d s M BP are s h o wn i n d a rk gre en an d p al e gr een. Th e co n f o rm at i o n o f s u cro s e cre at es a cav i t y t h at wo u l d n o rm al l y b e fi l l ed wi t h at o m s fro m t h e s eco n d gl u cos e u n i t o f m al t o s e, t o wh i ch t h ree ch ar g ed res i d u es (D1 4, K1 5, an d E1 1 1 ) wo u l d b e h yd ro g e n b o n d ed, as i l l u s t rat ed i n P an el B. Th e m u t at i o n s i n s M BP (p al e gr een ) co n v ert t h es e t h re e ch ar g ed r es i d u es t o n eu t ral res i d u es. 92

104 93

105 t h e t em p erat u r e fa ct o rs f o r t h e wi l d -t yp e co n fo rm at i o n are s i m i l ar t o t h e m ai n ch ai n av era g e, as i s t h e cas e wi t h op en, wi l d -t yp e M BP [ 5 ]. On t h i s bas i s, t h e o ccup an c y o f t h e wi l d t yp e co n fo rm at i o n i s es t i m at e d at 0.4, an d t h at o f t h e al t ern at e co n fo rm at i o n i s 0.6. Bo t h t h e op en an d cl o s ed co n fo rm at i o n s o f M BP a re i n v o l v ed i n m al t o s e t ra n s p o rt [ 8,1 4,1 5,1 6 ]. To u n d ers t an d wh y s M BP h as s u ch a co m p ro m i s ed ab i l i t y t o s t i m u l at e t h e M al FGK 2 ATP as e, w e co m p ared i t s s u rfa ce i n b o t h t h e op en an d cl o s ed co n fo rm at i o n s t o t h at o f wt M BP. Th e ch an ges i n s M BP n eces s ar y t o s up p o rt s u cro s e b i n d i n g req u i re o n l y s i d e ch ai n s ub s t i t u t i o n s, m o s t o f wh i ch are b u ri ed i n t h e s u ga r b i n d i n g s i t e an d n o t s u r face ac ces s i b l e i n t h e cl o s ed fo rm o f t h e p ro t ei n. As a res u l t t h e s u rf ac e m o rp h o l o g y o f cl o s ed s M BP i s v i rt u al l y u n al t ered f ro m cl o s ed wt M BP (Fi gu r e 3.5 A), wi t h o n l y a s l i gh t p ert u rb at i o n cau s ed b y t h e ex p o s u re o f a m et h yl g ro up o n D1 4 L (Fi gu r e 3.6 ). In co n t ras t t o t h e cl o s ed s t at e, t h e op en co n fo rm ati o n o f s M BP fu l l y ex p o s es al l 4 b i n d i n g s i t e m u t at i o n s t o t h e s o l ven t (Fi gu r e 3.5 B ) as w el l as t h e al t ern at e an d p a rt i a l l y d i s o rd er ed co n fo rm at i o n s fo r res i d u es 6 2 t o 6 9, cau s ed b y t h e W 6 2 Y m u t at i o n (Fi gu r e 3.4 ). To s u m m ari z e, o u r s t ru ct u ral an al ys i s fo u n d t h at i n t h e s u cro s e- b o u n d cl o s ed fo rm, s M BP cl o s el y m i m i cs t h e s u rfac e m o rp h o l o g y o f w t M BP, b u t op en u n l i gan d ed s M BP d i s p l a ys a d ras t i cal l y al t ered s u ga r- b i n d i n g s i t e. Altered interactions between open smbp and MalFGK 2 To i n v es t i gat e h o w t h e m u t at i o n s i n op en s M BP co u l d cau s e s u ch a d ras t i c d efect i n i t s ab i l i t y t o s t i m u l at e t h e M al FGK 2 ATP as e, w e r ep l aced wt M BP wi t h s M BP i n t h e cr ys t al s t ru ct u re o f M BP -M al F G K 2 t h at co rres p o n d s t o t h e t ran s i t i o n s t at e fo r ATP h yd ro l ys i s [ 7,8 ]. Th e b ackb o n e p o s i t i o n s o f s M BP fi t t h e M BP co m p o n en t o f t h e t rap p ed t ran s i t i o n s t at e t o an R M S D o f Å. 94

106 Fig 3.4 Main Chain Disorder in the Open Unliganded smbp Structure R es i d u es 6 2 t o 6 9 ad op t t wo d i ffer en t co n fo rm at i o ns i n t h e op en, u n l i gan d e d s t ru ct u re o f s M BP, wh i ch was r efi n e d t o 1.5 Å res o l u t i o n (Ta b l e 1 ). Th e m o l ecu l ar s u r face o f s M BP (ex cl u d i n g res i d u es 6 2 t o 6 9 ) i s s h o wn wi t h res i d u es 6 2 t o 6 9 i n cl u d ed as s t i ck m o d el s i n ei t h er (A) t h e co n fo rm at i o n res em b l i n g t h at ob s erv ed i n wtm BP, o r ( B) t h e al t e rn at e co n fo rm at i o n ob s erv ed i n s M BP. (C ) A s up erp o s i t i o n o f t h e t wo co n fo rm at i o n s ob s erv ed i n s M BP, al o n g wi t h 2 F O - F C el ect ro n d en s i t y c al cu l at ed f ro m p h as es ob t ai n ed th ro u gh a s i m u l at e d an n eal i n g p ro c ed u r e wi t h res i d u es 6 2 t o 6 9 o m i t t ed fro m t h e s t ru ct u re. No t e t h e el e ct ro n d en s i t y fo r Y6 2 i n t wo d i fferen t p o s i t i o n s. Th e A p o s i t i o n fo r Y6 2 co rr e s po n d s t o t h e p o s i t i o n o f W 6 2 i n wt M BP ; o ccup an c y o f t h e B p o s i t i o n req u i res an al t ern at e c o n fo rm at i o n fo r t h e m ai n ch ai n res i d u es. 95

107 96

108 Fig 3.5 Effect of Mutations on the Surface Properties of Open and Closed smbp Th e m o l ecu l ar s u rfa ces o f wt M BP (t an ) an d s M B P (p al e g re en ) a re co m p a red fo r b o t h t h e (A) cl o s ed an d ( B) op en co n fo rm at i o n s. In t h e cl o s ed co n fo rm at i o n, t h e s u r faces o f b o u n d m al t o s e an d s u cro s e are c o l o u red o ran g e an d d a rk gr een, r es p ect i v el y. Th e D1 4 L an d W 6 2 Y m u t at i o n s are v i s i b l e (red p at ch es ), b u t h av e v er y l i t t l e effect o n t h e acc es s i b l e s u rfac e o f s M BP co m p ared t o wt M BP. (B ) In t h e op en co n fo rm at i o n, al l o f t h e m u t at i o n s are v i s i b l e (red p at ch es ). In ad d i t i o n, t h e m ai n ch a i n res i d u es 6 2 t o 6 9 are p art i al l y d i s o rd ered ; t h e al t ern at e co n fo rm at i o n fo r t h es e r es i d u es i s s h o wn i n m ag en t a. C o o rd i n a t es fo r op en a n d cl o s ed w t M BP co rres p o n d t o 1 OM P [ 5] an d 1 ANF [ 2 4 ], res p ect i v el y. 97

109 98

110 Fig 3.6 Mutation D14L of smbp Ap art fro m t h e s u g ar l i g a n d (d ark gr een ), t h e o n l y s u rfac e ac ces s i b l e ch an g e b et we en cl o s ed wt M BP an d cl o s e d s M BP i s res i d u e D1 4 L (p al e gr een ). Th i s m u t an t res i d u e ex t en d s t h e h yd rop h ob i c s u rfa ce o f s M BP 2 Å fu rt h e r fro m t h e C A at o m o f D1 4 i n M B P (t an ). 99

111 100

112 Th e b i n d i n g o f s M BP t o t h e t ran s p o rt er t r an s m em b rane (M al FG ) d o m ai n s was n o t ob v i o u s l y co m p ro m i s ed acro s s t h e ex t eri o r s u r fac e of t h e b i n d i n g p ro t ei n, i n cl u d i n g co n t act s b et ween s M BP an d t h e M al F P 2 arm [ 1 7,1 8 ]. Ho wev er m u t at i o n s D1 4 L, W 6 2 Y an d E1 1 1 Y d i s rup t ed i n t eract i o n s o f t h e m al t o s e-b i n d i n g s i t e wi t h res i d u es o f M al G, wh i ch o c cup y t h e m al t o s e b i n d i n g s i t e i n t h e t ran s p o rt er t r an s i ti o n s t at e (Fi gu r e 3.7 ). Th e s e res i d u es co m p ri s e an i n v as i v e s t ru ct u r e k n o wn as t h e M al G P 3 s co o p l o op, n am ed fo r i t s p ro b ab l e ro l e i n ex cl u d i n g m al t o s e fro m t h e M BP m al t o s e-b i n d i n g s i t e [ 8]. D1 4 L cl as h es w i t h N2 5 4 o f M al G wh i l e W 6 2 Y an d E1 1 1 Y rem o v e s t ab i l i z i n g v an d er W aal s an d h yd ro g en b o n d i n t eract i o n s. In ad d i t i o n, t h e al t ern at e co n fo rm at i o n ad op t ed b y r e si d u es wi l l i n t erfer e wi t h M al G i n t eract i o n s. Al t o g et h er, t h e m u t at i o n s i n t h e op en co n fo rm at i o n o f s M BP wo u l d b e ex p ect ed t o d i s rup t i n t eract i o n s wi t h t h e M al G P 3 l o op i n t h e tran s i t i o n s t at e fo r ATP h yd ro l ys i s. In s u m m a r y, t h e m u t at i o n s i n s M BP h av e a d r as t i c eff ect o n i t s ab i l i t y t o s t i m u l at e M al FGK 2 ATP as e act i v i t y. S t ru ct u r al an al ys i s i n d i cat es t hat t h i s eff ect i s d u e t o a d i s rup t i o n o f i n t eract i o n s b et we en res i d u es o f M al G a n d t h e em p t y s u ga r b i n d i n g s i t e o f M BP as i t o ccu rs i n t h e t ran s i t i o n s t at e fo r ATP h yd ro l ys i s. Th e m a gn i t u d e o f t h i s eff ect s h o ws t h at t h es e i n t eract i o n s ar e cri t i cal f o r s t i m u l at i o n o f t h e M al FGK 2 ATP as e. 101

113 Fig 3.7 Interactions Between the MalFGK 2 P3 Loop and the smbp Ligand Binding Site Th e op en co n fo rm at i o n o f s M BP was s up e ri m p o s e d o n t o wt M BP i n t h e t ran s i t i o n s t at e s t ru ct u re o f t h e M BP -M a l FGK 2 co m p l ex (P DB- ID 2 R 6 G; [ 8]. Th e M al G P 3 s co op l o op co m p ri s i n g r es i d u es t o (c yan ) ex t en d i n t o t h e s u ga r b i n d i n g s i t e, m ak i n g co n t act s wi t h wt M BP res i d u es 1 4, 6 2, an d ( yel l o w). Th e co n t act s m ad e b et w een M al G an d t h e l i g an d b i n d i n g s i t e wo u l d b e a ff ect ed b y t h e m u t at i o n s i n s M BP. In ad d i t i o n, i n t er act i o n wi t h t h e M al G P 3 l o op wo u l d b e d i s rup t ed d u e t o d i s o rd er i n s M BP res i d u es 6 2 t o 6 9, as o u t l i n ed i n Fi gu r e

114 103

115 3.4 Discussion W e ob s erv ed t h at a s u c ro s e-b i n d i n g m u t an t o f M B P was ab l e t o s t i m u l at e t h e ATP as e act i v i t y o f M al FGK 2 wi t h ei t h er m al t o s e o r s u c ro s e p res en t as s ub s t r at e. T h e act i v at i o n was i n d i s t i n gu i s h ab l e b et we e n m al t o s e an d t h e n o n -p h ys io l o gi cal s ub s t rat e, s u c ro s e. Th e av ai l ab l e ev i d en ce s u g g es t s t h at s u cro s e i s u n ab l e t o i n t er act wi t h t h e m al t o s e b i n d i n g s i t e i n M al FGK 2. Fo r ex am p l e, i n t ran s p o rt as s a ys u s i n g M BP -i n d ep en dan t M al F GK 2 m u t an t s, s u cro s e was i n cap ab l e o f co m p et i t i v el y i n h i b i t i n g t h e t r an s p o rt o f m al t o s e [ 1 3 ] i n d i cat i ng t h at s ub s t rat e b i n d i n g s i t e o f M al F GK 2 h as l i t t l e, i f an y, affi n i t y fo r s u cro s e. Th i s can b e e x p l ai n ed u s i n g t h e M al FGK 2 s t ru ct u re i n co m p l ex wi t h m al t os e [ 8] : m o d el i n g s ucro s e i n t o t h e s am e p o s i t i o n as m al t o s e res u l t s i n cl as h es wi t h M al F res i d u es 3 8 3, an d 4 3 6, i n cl u d i n g s t eri c cl as h es wi t h b ackb o n e at o m s. S i n ce s u cro s e i s u n ab l e t o o c cup y t h e m al t o s e b i n d i n g s i t e o f M al F GK 2, t h e ob s erv at i o n t h at m al t o s e- an d s u cro s e -b o u n d s M B P h av e eq u al ab i l i t i es t o s t i m u l at e M al FGK 2 d em o n s t rat es t h at s p eci fi c b i n d i n g o f t h e ca rb o h yd rat e b y M al FGK 2 i s n o t i m p o rt an t fo r act i v at i o n o f i t s ATP as e. Th erefo re i t i s t h e s ub s tra t e i n d u ced co n fo rm at i o n a l ch an ge i n M BP, b u t n o t t h e i d en t i t y o f t h e s u b s t rat e i t s el f, t h at i s cri t i c al fo r s t i m u l at i o n o f t h e M al FGK 2 ATP as e. In p ri n ci p l e, ATP -d ep en d en t t ran s p o rt ers s h o u l d coup l e ATP h yd ro l ys i s t o act u al m o v em en t o f s ub s t rat e. Ou r res u l t s wi t h s M BP s h o w t h at d i rect i n t er act i o n s wi t h t h e s ub s t rat e are n o t r eq u i red fo r s t i m u l at i o n o f t h e M al FGK 2 A TP as e, an d t h er efo r e co u p l i n g o f ATP h yd ro l ys i s t o s ub s t rat e t r an s l o cat i o n m u s t d ep en d so l el y o n i n t er act i o n s b et ween M BP an d M al FGK 2. In t h i s re ga rd, t h e v er y s t ro n g d e fect i n t h e abi l i t y o f s M BP t o s t i m u l at e t h e M al FGK 2 ATP as e i n d i cat es t h at a c ri t i cal i n t eract i o n b et w ee n M BP an d M al F GK 2 h a s b een d i s rup t ed b y t h e m u t at i o n s. Bo t h t h e op en an d cl o s ed co n fo rm at i o n s o f M BP i n t eract wi t h M al FGK 2 d u ri n g t h e cat al yt i c c ycl e [ 3,7,8,1 5 ]. Th e s u rfa ce o f cl o s ed s M BP i s al m o s t i d en t i cal t o t h at o f wt M BP, 104

116 s u g ges t i n g t h at t h i s co n f o rm at i o n i s n o t res p o n s i b l e fo r t h e r ed u ced ab i l i t y o f s M BP t o s t i m u l at e t h e M al FGK 2. In f act, L1 4 (D1 4 i n wt M BP ) i s t h e o n l y m u t an t r es i d u e t h a t cau s es a ch an ge i n t h e ex p o s ed s u rfac e o f cl o s ed s M BP an d co u l d t h e refo r e al t er i n t e ract i o n s wi t h M al FGK 2. Al t h o u gh fu n ct i o n al g en e t i c s creen s h av e d em o n s t rated t h at t h e r e gi o n aro u n d res i d u e 1 4 i s i m p o rt an t fo r t h e i n t era ct i o n o f M BP an d M al F G K 2 [ 1 9,2 0], t h e o n l y ch an ge fo u n d at r es i d u e 1 4 i n t h e gen et i c s cr een s w a s a m u t at i o n t o t yro s i n e, a m u ch l ar ge r res i d u e t h a t can n o t b e b u ri ed i n t h e l i gan d -b o u n d co n fo r m at i o n o f M BP, an d wo u l d t h erefo r e p ro d u c e a l a r ge ch an g e i n t h e s u rfac e o f t h e cl o s ed co n fo rm at i o n. In co n t r as t, t he D1 4 L m u t at i o n i s m o s t ly b u ri ed an d al m o s t i s o s t eri c, res u l t i n g i n o n l y a v er y s m al l ch an ge i n t h e s u rfac e, n am el y a 2 Å ex t en s i o n o f an ex i s t i n g h yd rop h ob i c p at ch (Fi gu r e 3.6 ). Th ere fo r e, t h e s m al l eff ect o f t h e D1 4 L m u t at i o n o n t h e s u rfac e o f cl o s ed s M BP d o es n o t p ro v i d e a co n v i n ci ng ex p l an at i o n fo r t h e p ro fo u n d ef fect o f t h e m u t at i o n s o n t h e ab i l i t y o f s M BP t o s t i m u l at e M al FGK 2. Th e s u rfa ce o f op en s M B P, o n t h e o t h er h an d, i s d ras t i cal l y al t er ed b y t h e e x p o s u re o f m u t an t res i d u es i n t h e s u ga r-b i n d i n g s i t e an d t h e c reat i o n o f an are a o f co n f o rm at i o n al i n s t ab i l i t y d u e t o t h e W 6 2 Y m u t at i o n. On t h i s b as i s, t h e p ro f o un d d efect i n s M BP i s m o s t l i k el y d u e t o a d i s rup t i o n o f i n t eract i o n s b et ween t h e op en, rat h er t h an t h e cl o s ed, co n fo rm at i o n o f M BP. Th i s co n cl u s i o n i s co n s i s t en t wi t h an i m p o rt an t ro l e fo r op en M BP i n s t ab i l i z at i o n o f t h e t ran s i t i o n s t at e fo r ATP h yd ro l ys i s [ 8,1 5 ]. In fact, t h e red u c ed a ct i v at i o n o f M al FGK 2 ATP a s e b y s M BP co i n ci d es wi t h a d i s rup t i o n o f i n t eract i o n s b et w een s M BP l i gan d b i n d i n g s i t e res i d u es an d t h e i n v as i v e M al G P 3 l o op o f M al FGK 2 [ 8 ]. Th es e i n teract i o n s a re o n l y p o s s i b l e o n ce M BP ad op t s t h e o p en co n fo rm at i o n an d m al t o s e h as v ac at ed t h e M BP s u g ar b i n d i n g s i t e t o en t er M al FGK 2. Th e i n t eract i o n s b et w een t h e M al G P 3 l o op an d t h e l i g an d b i n d i n g s i t e o f M BP ap pear t o p l a y a c ri t i cal r o l e i n t ran s p o rt co up l i n g b y al l o wi n g h yd ro l ys i s o f ATP o n l y o n c e t he l i g an d h as t ran s l o c a t ed fro m t h e b i n d i n g 105

117 s i t e i n M BP t o t h e M al F GK 2 b i n d i n g s i t e. A ro l e fo r t h e M al G P 3 l o op i n en er get i c co up l i n g i s co n s i s t en t wi t h i t s p o si t i o n i n M al FGK 2 (Fi gu re 3.8 ). T h e P 3 l o op i s co n n ect ed t o M al G h el i ces 1 5 an d 1 6, wh i ch ex t en d s fro m t h e s co op l o op t o t h e M al G C -t erm i n u s, l o cat ed i n a h yd ro gen b o n d n et wo r k eq u i d i s t an t b et ween t h e t wo ATP b i n d i n g s i t e s o f M al K 2. Ou r d at a i n d i cat e t h at i n ad d i t i o n t o ex t ract i n g m al t o s e fro m t h e M BP s u g ar b i n d i n g c l eft [ 8 ] i nt eract i o n s b et w een t h e M al G P 3 l o op an d M BP al s o p l a y a d i rect ro l e i n p ro m o t i n g ATP h yd ro l ys i s. Th es e i n t eract i o n s d o n o t d ep en d o n t h e s p eci fi c ch em i cal i d en t i t y o f t h e s ub s t rat e, an d t h er efo r e a s i m i l ar m ech an i s m m i gh t b e op erat i v e i n m u l t i - d ru g ex p o rt ers an d o t h er ABC t ran s p o rt ers t h at co upl e ATP h yd ro l ys i s t o t h e t ran s p o rt o f d i v e rs e s ub s t rat es. 106

118 Fig 3.8 Structural Basis for Communication Between the MalG P3 Loop and ATP Binding Cassettes Th e M BP -M al FGK 2 t ran s i t i o n s t at e co m p l ex (P DB- ID 2 R 6 G)[ 8 ] i s s h o wn. As d et ai l ed i n Fi gu re 5, M al G res i d u es t o (c ya n ) m ak e co n t act s wi t h t h e s u ga r b i n d i n g s i t e o f M BP (p al e g reen ). Th es e co n t a ct s are o n l y p o s s i b l e wh en m al t o s e i s ab s en t, h en c e t h e t erm s co op l o op was u s ed t o a s si gn a fu n ct i o n t h i s regi o n o f M al G [ 8 ]. R es i d u es t h at are m u t at ed i n s M BP are s h o wn i n gre en C P K rep r es en t a t i o n. Th e l o op i s co n n ect ed wi t h t h e M a l K 2 ATP b i n d i n g s i t es (re d ) v i a t h e M al G C -t erm i n u s (vi o l et ). 107

119 108

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121 k i n et i cs o f b i n d i n g p ro t ei n d ep en d en t t ran s p o rt s ys t em s rev eal s t h at b o t h t h e s ub s t rat e l o ad ed an d u n l o ad ed b i n d i n g p ro t ei n s i n t eract wi t h t h e m em b ran e co m p o n en t s. J. Theor. Biol. 172: Dau s, M. L., Gro t e, M., a n d S ch n ei d er, E. ( ) Th e M al F P 2 l o op o f t h e ATP - b i n d i n g cas s et t e t ran s p o rt er M al F GK2 fro m Es ch e ri chi a co l i an d S al m o n el l a en t eri ca s ero v a r t yp h i m u ri u m i n t eract s wi t h m al t o s e b i n d i n g p ro t ei n (M al E ) t h ro u gh o u t t h e c at al yt i c c yc l e. J Bacteriol 191: Gro t e, M., P o l yh a ch, Y., J es ch k e, G., S t ei n h o ff, H. J., S ch n ei d er, E., an d Bo rd i gn o n, E. ( ) Tran s m em b ran e s i gn al l i n g i n th e m al t o s e ABC t ran s p o rt er M al FGK2 - E: p eri p l as m i c M al F -P 2 l o op co m m u n i cat es sub s t rat e av ai l ab i l i t y t o t h e ATP -b o u n d M al K d i m er. J Biol Chem 284: Ho r, L., an d S h u m an, H. ( ) Two m o d es o f l i gan d b i n d i n g i n m al t o s e-b i n d i n g p ro t ei n o f Es ch e ri ch i a co l i. Fu n ct i o n al s i gn i fi c an c e i n act i v e t ran s p o rt. J. Mol. Biol. 233: Trep t o w, N., an d S h u m a n, H. ( ) Al l el e -s p eci fi c m al e m u t at i o n s t h at re s t o re n t eract i o n s b et w een m al t o s e-b i n d i n g p ro t ei n an d t h e i n n er-m em b r an e co m p o n en t s o f t h e m al t o s e t ran s p o rt s ys t em. J. Mol. Biol. 202: Brü n ge r, A. T., Ad am s, P. D., C l o re, G. M., De Lan o, W. L., Gro s, P., Gro s s e- Ku n s t l ev e, R. W., J i an g, J. S., Ku sz ews k i, J., Ni l ges, M., P an n u, N. S., R ea d, R. J., Ri ce, L. M., S i m o n s o n, T., an d W arren, G. L. ( ) C r ys t al l o gr ap h y & NM R s ys t em s : A n ew s o ft war e s u i t e fo r m acro m o l e cu l a r s t ru ct u re d et erm i n at i o n. Acta Crystallogr. D Biol. Crystallogr. 54: Em s l e y, P., an d C o wt an, K. ( ) C o o t : m o d el -bu i l d i n g t o o l s fo r m o l ecu l ar gr ap h i cs. Acta Crystallogr D Biol Crystallogr 60: De Lan o, W. ( ) The PyMOL User's Manual 2 4. Qu i o ch o, F. A., S p u rl i n o, J. C., an d R o d s et h, L. E. ( ) Ex t en s i v e feat u r e s o f t i gh t o l i go s a cch a ri d e b i n d i n g rev eal ed i n h i gh - res ol u t i o n s t ru ct u res o f t h e m al t o d ex t ri n t ran s p o rt / ch em o s en s o r y re cep t o r. Structure 5 : Tel m er, P.G., S h i l t o n, B.H., ( ) S t ru ct u ral s t u d i es o f an en gi n e ered z i n c b i o s en s o r rev e al an u n an t i ci p at ed m o d e o f z i n c b i n di n g, J Mol Biol 354:

122 Chapter 4 Activation of the Maltose Transporter by Cysteine Stabilized MBP Mutants 4.1 Introduction The maltose transporter (MalFGK2) of Escherichia coli efficiently couples the hydrolysis of ATP to the internalization of maltose. This coupling is enforced by the prevention of futile ATP hydrolysis when the transporter is in a resting state and has not been activated by its primary substrate receptor, the maltose binding protein (MBP). Under the influence of maltose liganded MBP this autoinhibition is reversed and rapid ATP hydrolysis is coupled to maltose transport [1,2,3]. MBP binds maltose and other short maltodextrins at the interface between its two domains. On the binding of maltose, interactions within the maltose-binding site cause these domains to rotate about a flexible hinge region and close around the bound ligand. This results in a shift in conformation from a stable open resting state to a closed state that has been likened to the snapping shut of a venus flytrap [4,5]. The closed conformation of MBP is known to play a major role in transporter stimulus as only those MBP ligands that induce domain closure are transported [3]. Additionally, MBP residues identified as significant for stimulus cluster together in the closed, but not open, conformation [6]. The transporter is thought to act by an alternating access model, in which conformational changes during the ATPase catalytic cycle alternately expose an internal binding site to the periplasm, where substrate can be collected, and to the cytoplasm, where it can dissociate [7]. It has been demonstrated that during these conformational changes the transporter interacts with both the closed and open forms of MBP which could suggest that both forms play functional roles [8,9,10]. 111

123 Previous analysis has supported this theory by showing that the activity of MBP is tied to the stability of both its dominant conformers. Destabilizing the unliganded open form of MBP, and thus shifting the protein towards closed unliganded forms, decreased the ATPase observed with unliganded MBP [11]. This indicates that the stability of the open form, or the ability of the closed form to convert to the open, plays a role in ATPase stimulus. The open conformation is likely responsible for the activation of ATP hydrolysis seen with unliganded MBP, suggesting that the interaction between open MBP and the transporter, seen in the ATPase transition state, promotes ATP hydrolysis [8]. That ATPase stimulus in the absence of maltose was not consistent with causation by transiently closed MBP may further be an indication that the stability of the closed form is a factor in activity [12]. These findings justify a deeper investigation of the separate functional roles of the closed and open forms of MBP, however previous investigations have been complicated by the conformational flexibility of MBP. MBP exists in an equilibrium between its most stable forms, ligand bound closed and unliganded open, and more transient forms such as unliganded semi-closed and ligand-bound open conformations [13,14]. This equilibrium, and an understanding that closed MBP is opened by the transporter during the transport cycle, obscures the role of each MBP conformation. The intent in this study was to better de-convolute the separate abilities of the closed-liganded and the unliganded-open forms of MBP to support ATP hydrolysis by the transporter. To this end we have exploited the known structures of MBP to introduce cysteine mutations and cross-links predicted to stabilize the 112

124 open or closed forms of MBP. These interventions were intended to shift or abolish the equilibrium between open and closed MBP, producing binding proteins that favor a single conformation. The transporter has additionally been extracted from the lipid bilayer and solubilized using a mild detergent. This treatment is known to disrupt the transporters normal auto-inhibiton behavior and result in a much higher constitutive ATPase activity [15]. If both forms of MBP play a role in transport, then this disruption in regulation may differently impact the ability of the open and closed forms to stimulate ATP hydrolysis, enabling their individual contributions to be better determined. Here we determine that a closed form of MBP is sufficient to activate substantial, although not maximal, ATPase activity in vitro. However, when the relative stabilities of differing transporter conformations are altered by detergent solubilization, the ability of the closed conformation of MBP to stimulate the MalFGK2 ATPase is relatively small compared to that of unliganded open MBP. This is the opposite of the result seen from the membrane bound system. This suggests that the role played by the closed form is diminished in importance, while that of the open form is increased for detergent solubilized MalFGK2. This supports our hypothesis that both conformations play functional roles in hydrolysis; with the liganded-closed state mediating a conversion of the transporter to its catalytic conformation and the unliganded-open state stabilizing the catalytic state to promote ATPase. These results also indicate that the conformational stability, or the availability, of those transporter conformations targeted by the closed and open 113

125 forms of MBP is sensitive to packing interactions between the transmembrane regions and lipid bilayer. Freeing the complex from the membrane simultaneously makes the transporter more available to productively interact with open MBP and less available to closed MBP. We additionally find that a mutant MBP, stabilized in the open conformation by a cysteine coupled cross-linker, stimulates a high degree of ATPase activity from the transporter even in the absence of maltose. This uncoupling phenotype offers insights into the conformational changes of the transporter following hydrolysis, as it leaves the ATPase catalytic state and it reverts to the resting state. 114

126 4.2 Materials and Methods Cloning, Growth and Purification of Binding Protein Mutants Cloning of MBP-Closed (MBP-C), MBP-Open (MBP-O) and MBP-O95/171 A previously identified mutant of MBP was reproduced, with cysteine mutations introduced at positions G69C and S337C (MBP-C) of the otherwise cysteineless MBP [16]. Two additional mutants were engineered, MBP-O and MBP- O95/171, with cysteines introduced at positions G101C/G174C and D95C/Y171C respectively. These mutations were cloned into plasmid pproex-hta [12,17] containing a previously cloned sequence for wtmbp lacking a periplasmic export tag but with a TEV-protease cleavable hexahistidine tag. These mutations were incorporated by two rounds of mutagenic PCR utilizing the stated complementary primer pairs (Table 4.1). Expression of MBP Mutants MBP-C was expressed from pproex-hta in a BL21 (DE3) background by induction with IPTG as previously performed for other binding protein mutations [12,17]. Cells were lysed by passage through a French Pressure cell and expressed protein extracted by Ni 2+ affinity, followed by cleavage of the histidine tag and repeat passage across a Ni 2+ -loaded Chelating Sepharose column as previously described [12,17]. MBP-O, MBP-O95/171 and wtmbp were expressed and extracted as described for MBP-C. wtmbp was additionally unfolded by serial dialysis against 50 mm Tris- Cl ph8.0, 100 mm NaCl, 6 M guanidine-hcl to reduce maltose contamination, then refolded and purified as previously reported [12]. 115

127 Table 4.1 Mutagenic Primers for MBP Mutants Mutant Mutation Primer Sequence (5'->3') MBP-C69/337 G69C CGACCGCTTTGGTTGCTACGCTCAATCTGGCC S337C CCCGCAGATGTGCGCTTTCTGGTATGCCG MBP-O 101/174 G101C CGTACGTTACAACTGCAAGCTGATTGCTTACC G174C CGTTCAAGTATGAAAACTGCAAGTACGACATTAAAGACG MBP-O95/171 D95C CGTTTACCTGGTGTGCCGTACGTTAC Y171C GGTTATGCGTTCAAGTGTGAAAACGG 116

128 Oxidation and Cross-Linking of Binding Protein Mutants Purified MBP-C and MBP-O mutants were dialyzed in 50 mm NaH2PO4 ph 7.0, 100 mm NaCl, 1 mm TCEP to reduce mixed disulfides that may have formed during lysis or TEV digestion. After dialysis in 50 mm NaH2PO4 ph 7.0, 100 mm NaCl to remove reducing agent both proteins were left to incubate for 2 days with 10 µm CuCl2; MBP-C with 10 µm of maltose and MBP-O mutants without substrate. Oxidized MBP-C was purified by affinity chromatography on immobilized amylose (New England Biolabs) and collected from the non-binding flow-through fraction. MBP-C was then concentrated and purified by size exclusion chromatography on a 60 x 2.6 cm Superdex 200 (GE Healthcare) column equilibrated with 50 mm Tris-Cl ph 8.0, 100 mm NaCl. MBP-C was concentrated and frozen in aliquots at -80 C for future use. CuCl2 failed to oxidize MBP-O and instead the protein was cross-linked by reaction with 1,2-Bis(Maleimido)ethane (BMOE). The protein was dialyzed in 20mM Hepes ph 7, 0.1 M NaCl and diluted to 4 mg/ml. BMOE modification was accomplished by incubating the protein at room temperature and adding a ½ molar equivalent of BMOE (dissolved in DMSO) up to 10 times at half hour intervals. The cross-linking reaction was left to complete at 4 C overnight. MBP-O was then concentrated and purified on a 60 x 2.6 cm Superdex 200 (GE Healthcare) column equilibrated with 50 mm Tris-Cl ph 8.0, 100 mm NaCl. MBP-O95/171 was expressed, modified and purified in the same way as MBP-O. Following modification and purification, both proteins were concentrated and flash frozen at -80 C. 117

129 Aliquots of these frozen proteins were observed to be >99% pure by SDS- PAGE (appendix 1). Analysis of Protein oxidation and Cross-linking Oxidized MBP-C was analyzed by SDS-PAGE with samples prepared in a loading buffer lacking reducing agent. A mobility shift on non-reducing SDS-PAGE was seen, like that observed by Zhang et al., indicating disulfide formation [16]. Samples were additionally analyzed by SDS-PAGE using loading buffer without reducing agent, but containing 15 µm fluorescein maleimide (FM) and visualized by fluorescence on a UV table. This demonstrated the blocking of free sulfhydrils by a total loss of fluorescence labeling. Analytical samples of MBP-O and MBP-O95/171 were removed at 30 minute intervals during BMOE cross-linking and further reaction in the sample was arrested by the addition of excess FM. An additional sample was collected following over-night incubation of the BMOE reaction. All BMOE reaction samples were analyzed by SDS-PAGE and the degree of FM labeling was visualized by fluorescence on a UV table. The blocking of free sulfhydryls was observed as a reduction in fluoresce in progressing time-point samples. Mass spectrometry was performed on oxidized and modified proteins using an Applied Biosystems 4700 Proteomics Analyzer MALDI-TOF-TOF in linear mode. Prior to mass spectrometry all proteins were serially dialyzed against deionized water. The mass of the modified proteins was consistent with the covalent attachment of a single BMOE molecule. Note that when MBP-O was modified using 118

130 an excess concentration of BMOE, mass analysis indicated dual modification of the protein, with a separate BMOE linked to each cysteine. Measurement of Maltose Binding by Intrinsic Tryptophan Fluorescence Quenching Several tryptophan residues in wtmbp undergo significant changes in their local environments on maltose binding and MBP domain closure that, in aggregate, result in a partial quenching of fluorescence. The affinity of MBP mutants for maltose was therefore measured by intrinsic tryptophan fluorescence quenching. Fluorescence measurements of wtmbp, MBP-C and MBP-O were taken with a Horiba-Jobin Yvon Florolog-3 spectrofluorimeter. Proteins were diluted into 50 mm Tris-Cl ph 8.0, 100 mm KCl, 10 mm MgCl2 and excited at 280 nm. Fluorescence emission was measured at 347nm. Maltose was added with rapid stirring at 2- minute intervals and the decrease in fluorescence caused by maltose binding was recorded as a percentage of initial fluorescence [18]. KD values for wtmbp and MBP- O were derived using GraphPad Prism version 4.0b to fit data by non-linear regression to a Michaelis-Menten curve. Small Angle X-ray Scattering Analysis of Binding Protein Mutants Protein samples were thawed and aggregate was removed by size exclusion chromatography on a 60 x 2.6cm Superdex 200 (GE Healthcare) column equilibrated with 20 mm Tris-Cl ph 7, 100 mm NaCl, 4 % glycerol, 1 µm sodium azide. Small angle x-ray scattering (SAXS) was performed at the BioCAT beam line of the Argonne national labs Advanced Photon Source. Data collection and 119

131 processing were performed as previously described [12], and the radius of gyration was calculated with the program GNOM [19]. Preparation of Proteoliposomes and Detergent Solubilized Transporter Expression of MalFGK2 MalFGK2 was expressed in an AD126 background co-transformed with plasmids pfg23 and pkj [20]. The transmembrane subunits MalF and MalG were expressed from plasmid pfg23 while MalK, with a C terminal hexahistidine tag, was expressed from plasmid pkj [20]. A single colony was picked and grown for 6 hours in 5mL LB media as a starter culture under the selection of 100 µg/ml ampicillin (selective for pfg23), 50 µg/ml chloramphenicol (selective for pkj), and 50 µg/ml kanamycin (selective for a LacIq Tn5 transposon in AD126). The starter culture was used to inoculate fresh 2xYT media at a dilution of 1:1,000,000 with the same antibiotic selection. These cultures were grown to an OD600 of 0.4, induced with 10µg/mL IPTG and grown for 8 hours at 27 C. Cells were harvested by centrifugation, re-suspended in 20mM Hepes ph 7, 100mM NaCl, 5% Glycerol and flash frozen in liquid nitrogen for storage at -80 C. Preparation of Membranes Frozen cells were thawed, augmented by the addition of 1µM PMSF and lysed by 2 passes through a French pressure cell at 20,000x p.s.i. Cell debris was pelleted by centrifugation at 10,000 g for 30 minutes. Cell membranes were then pelleted by ultracentrifugation at 100,000 g for 1 hour. Membranes were resuspended and washed twice in 20mM Hepes ph 7.0, 5mM MgCl2, 200mM sucrose, then 120

132 resuspended a final time in 20mM Tris-Cl, ph 7.5, 5mM MgCl2, 25 mm KCl, flash frozen and stored at -80 C for future use. Solubilization of Membrane Proteins and Proteoliposome Reconstitution Thawed membranes were pelleted at 100,000 g for 1 hour and re-suspended at 5 mg/ml in 50 mm Tris-Cl ph 7.0, 10 mm MgCl2, 1.0 % dodecyl-maltoside. This solution was incubated on ice for 30 minutes while protein was solubilized. Insoluble material was removed by another centrifugation step at 100,000 g for 1 hour. Detergent solubilized MalFGK2 was purified by nickel affinity chromatography on a Ni 2+ charged, 30 ml, chelating Sepharose chromatography column equilibrated with 20mM Tris-Cl ph 7.0, 10mM MgCl2, 10% glycerol, 0.01% dodecyl-maltoside (Buffer A). The protein was washed with Buffer A and eluted in Buffer A with 250mM imidazole. The eluted protein was dialyzed over night against 20mM Tris-Cl ph 7.0, 10mM MgCl2, 10% glycerol, 0.01% dodecyl-maltoside to remove the imidazole. One half of this purified transporter solution was flash frozen as detergent solubilized transporter. The remaining protein was augmented with 1% B-octylglucoside, incubated for 30 minutes, then reconstituted into previously prepared lipsomes by dilution, as previously performed [12]. Liposomes were prepared by the Bangham method as previously described [12,21]. Reconstituted proteoliposomes, with incorporated MalFGK2, were flash frozen and stored at -80 C for future use. The presence of MalF and MalK in the final preparations of proteoliposomes and detergent solubilized transporter was confirmed by SDS-Page and Coomassie stain (appendix 2). 121

133 Assay of ATPase Activity ATPase assays were performed as previously described [12]. Briefly, proteoliposomes were added to a concentration of 0.1mg/mL in 342 µl of 50mM Tris-Cl ph 8.0, 100mM KCl, 10mM MgCl2, in the presence or absence of maltose and purified binding protein. The reaction was started by the addition of 18µL of 80µM ATP in the same buffer. 60µL samples were removed at time 0, 5, 10, 15 and 20- minute time intervals and arrested with 30µL of 10% SDS solution. Once the reaction was complete, all samples were measured by the addition of 50µL of Molybdate color-reagent (1.6% Ammonium Molybdate, 5% Ferrous Sulfate- Heptahydrate, 2M Sulfuric Acid) and measurement at 740nM on a Varian Cary 100Bio UV-Vis spectrophotometer. Absorbance measurements were converted to phosphate concentrations using a Na2PO4 standard curve ranging from 0 to 1mM phosphate. Phosphate concentrations for each time point were analyzed by linear regression and normalized against the mass of MalFGK2 used to obtain the rate of ATP hydrolysis. 122

134 4.3 Results Expression of a Locked closed Maltose Binding Protein MBP undergoes well-characterized domain rotations from open to closed on the binding of maltose, closing the maltose binding site and partially burying the substrate [5]. Previous studies suggest that both open and closed conformations, as well as dynamic domain rotations might play roles in the activation of the transporter by MBP [10,11,12,16]. However, analysis of the respective roles of closed and open MBP can be complicated by the flexibility of the protein and interchange between these two conformations. In principle it is possible to incorporate mutations into MBP to alter substrate binding or domain rotation [11,22]. Here a mutant form of MBP has been used, which is predicted to be perpetually in the closed conformation. This mutant enables study of the role of closed MBP, in transporter activation, in isolation from the open form and competing conformational changes. This protein enables us to observe the ability of the closed form of MBP to activate ATP hydrolysis. A disulfide containing MBP mutant was reproduced from the studies of Zhang et al. [16]. The mutant was engineered with cysteine substitutions at positions G69 and S337 (Fig. 4.1). Crystal structures of wtmbp indicate that these mutations are near the maltose-binding cleft and are proximal in the maltose-bound closed conformation, with sulfur atoms 4.1 Å apart. However, in the open state the sulfur atoms are separated by 6.5 Å (Fig. 4.1). This separation is only compatible with a disulfide bond in the closed conformation, and should hold the protein in, or strongly favor, the closed conformation. The characterization and phenotype 123

135 Fig 4.1 MBP-C Cysteine Mutations. The positions and spacing of MBP-C cysteines G69C and S337C are shown (yellow) modeled onto X-ray crystal structures of the open and closed conformations. The distances shown correspond to the separation of sulphur atoms in a hypothetical cystine. The maltose-binding site is shown as a yellow wedge, with bound maltose (green and red). PDB Code 2R6G[8], 1ANF[4] 124

136 125

137 observed by Zhang et al. was consistent with this prediction [16]. In this study we refer to the disulfide constraining mutant MBP G69C/S337C as MBP-closed (MBP- C). At high levels of expression in-vivo, MBP-C is known to inhibit maltose transport in a dominant negative manner. This phenotype is reversed by reducing reagent, which indicates that it is caused by the disulfide bond [16]. Inhibition is likely a result of competition between MBP-C and wtmbp for binding sites on the maltose transporter. Although its dominant negative phenotype indicates that MBP- C cannot support maltose transport, the degree to which it can induce or inhibit ATP hydrolysis is not known. For that reason MBP-C was cloned, purified and oxidized in the presence of maltose. ATPase stimulation by MBP-C in proteoliposomes Wild-type maltose transporter (MalFGK2) was purified and reconstituted into proteoliposomes (PLS) made from E. coli phospholipids. Proteoliposomes form an environment for MalFGK2 similar to the native membrane, but are ideal for in vitro experimentation and have been widely used for this reason [23,24]. The successful incorporation of the transporter into finished proteoliposomes was confirmed by SDS-Page and Coomassie stain (appendix 2). With MBP-C prepared and oxidized we examined its ability to activate the maltose transporter. MBP-C was incubated at 37 C with PLS containing MalFGK2 and the resulting level of ATP hydrolysis was measured. Purified MalFGK2 displays a low level of basal ATPase in liposomes. As previously reported, in the absence of 126

138 maltose wtmbp activates a small level of ATPase above this background activity. When wtmbp is added with saturating maltose a large increase in activity is seen, which is dependent on binding protein concentration (Fig 4.2A). MBP-C was observed to activate the transporter to a significantly higher level than wtmbp, but far less than the fully activated system with wtmbp-maltose (Fig. 4.2B). The addition of maltose had no effect on the level of ATPase stimulus by MBP-C, although the rate was increased by the addition of maltose and DTT (data not shown). Given its dominant negative effects on maltose transport in vivo, we were surprised by the ability of MBP-C to stimulate ATPase activity. Further, MBP-C does not support growth on minimal maltose, which indicates that it cannot support significant maltose transport, but we observed that it can activate more than 25% of the maximal ATPase rate [16](Fig. 4.2). Given these unexpected results I explored the ability of MBP-C to stimulate detergent solubilized MalFGK2. ATPase stimulation of detergent uncoupled MalFGK2 by MBP-C and wtmbp The maltose transporter is most commonly studied in proteoliposomes because of the similarity of this system to the transporter s cellular environment. When extracted from the membrane and stabilized in solution by a mild detergent the transporter acquires a very high basal rate of ATP hydrolysis, independent of the presence of MBP [15,25]. This unregulated phenotype is reversible if the transporter is reconstituted into a membrane. This suggests that important features 127

139 Fig 4.2 ATPase Stimulus by MBP-C in Proteoliposomes. Purified MalFGK2 was reconstituted into proteoliposomes and incubated at 37 C with (A.) wtmbp, wtmbp with maltose (5mM) and (B.) MBP-C. The production of free phosphate was measured at 5-minute time intervals as described in Materials and Methods. The rate of ATP hydrolysis was calculated from these data using linear regression and a phosphate standard curve before being plotted for each binding protein concentration. 128

140 129

141 of the transporter catalytic cycle are dependent on membrane interactions, and altered by detergent solubilization. We hypothesized that disruption by detergent might differently impact the transporter conformations targeted by the closed and open forms of MBP. We therefore prepared MalFGK2 as per our previous experiments, but rather than reconstituting the transporter into membranes, left it solubilized by the mild detergent n-dodecyl-β-d-maltoside (DDM). We incubated purified wtmbp and MBP-C with the detergent stabilized transporter at 37 C and measured ATP hydrolysis as previously described. As reported elsewhere the transporter shows a much higher level of basal unregulated activity in the detergent soluble phase than in PLS and can be further stimulated by the addition of MBP and maltose (Fig. 4.3A) [15,25]. The addition of MBP-C resulted in a much smaller activation of ATP hydrolysis than wtmbp-maltose (Fig. 4.3B). Closed conformation MBP is therefore still relevant to transporter activation in detergent, although MBP-C stimulates a smaller fold-wise increase in activity over the basal rate because of the higher basal activity of the solubilized transporter. This suggests that the transporter conformation normally stabilized or complemented by closed MBP has been rendered less relevant in the detergent uncoupled system, although it is not removed from the catalytic cycle. This low-level activation in detergent by MBP-C is far more intriguing when contrasted with the surprising activation by unliganded wtmbp. Unlike in PLS, where a low level of ATP hydrolysis is observed, unliganded wtmbp activates a high level of hydrolysis from DDM solubilized transporter; more than MBP-C or wtmbp- 130

142 Fig 4.3 ATPase Stimulus by MBP-C of Detergent Solubilized MalFGK2. Purified MalFGK2 was extracted from the cell membrane and stabilized in 50 mm Tris ph7, 4mM MgCl2, 5% glycerol, 0.01% dodecyl-maltoside [15]. The resulting uncoupled transporter was incubated at 37 C with (A.) wtmbp, wtmbp with maltose(5mm) and (B.) MBP-C. The resulting rate of ATP hydrolysis was calculated as described in Fig

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144 maltose (Fig. 4.3A). This result may not have been previously reported because activation by apo-wtmbp was not thought to be informative, but shows that detergent solubilization has made the transporter more receptive to the open resting state of wtmbp. Concerns that these data might be explained by an ability of DDM to mimic maltose proved unfounded as intrinsic tyrosine fluorescence showed no binding of DDM to wtmbp and ATPase experiments demonstrated that DDM did not increase the ability of wtmbp to activate ATPase in PLS (data not shown). While in liposomes it appears the closed form of MBP, as represented by MBP-C, is more activating than open unliganded wtmbp, in the detergent solubilized system the relationship has reversed and wtmbp activates higher levels of ATPase than MBP-C. Characterization of MBP-C With such unexpected results observed in ATPase studies, it was necessary to better characterize how disulfide bond formation has altered the characteristics of MBP-C from the wild type. Due to the similar bond chemistries of amylose and maltodextrins, wtmbp shows substantial affinity for amylose and will bind to immobilized amylose affinity resin [26]. To characterize the effect of the interdomain disulfide on the activity of MBP-C we performed amylose chromatography. Unlike wtmbp, oxidized MBP-C was not retained by amylose, however binding was restored by reduction with DTT. Further, we measured the intrinsic tryptophan fluorescence of MBP-C as it was titrated with maltose. Unlike wtmbp, MBP-C shows 133

145 Fig 4.4 Maltose Binding Measured by Intrinsic Fluorescence Quenching Intrinsic Tryptophan Fluorescence quenching by maltose was measured for wtmbp, MBP-C and MBP-O in a Horiba-Jobin-Yvion Fluorolog-3 fluorimeter [18]. Proteins were excited at 280nm, and fluorescence was measured at 347nm. For each protein: (A) The reduction in fluorescence is shown at increasing maltose. (B) The reduction in fluorescence is expressed as a percentage of initial fluorescence, with Michaelis-Menten saturation curve. 134

146 135

147 little change in fluorescence with the addition of maltose (Fig. 4.4). Together these results suggest that MBP-C has no capacity to bind additional substrate. Samples of oxidized MBP-C were further analyzed by Small Angle X-ray scattering to observe the in-solution conformational changes brought on by the addition of maltose. This analysis showed a radius of gyration in the absence of maltose of 21.8 Å. This is consistent to the measured radius of maltose saturated wtmbp, 21.8 Å, suggesting that MBP-C is in the closed conformation. The addition of saturating maltose (5 mm) resulted in a virtually unchanged radius of gyration of 21.9 Å (Table 4.4). This was consistent with intensity difference curves of the protein s total SAXS spectrum with and without maltose, which demonstrate minimal change in the spectra of MBP-C on the addition of maltose (Fig. 4.5). These results indicate a significant change in the binding characteristics of MBP-C from wild type and suggest that our reproduction of the disulfide locked MBP mutant of Zhang et al was successful. The protein is perpetually locked in the closed conformation, likely with a permanently resident molecule of maltose occupying its binding site [16]. The increased ATPase stimulated in PLS, as compared to wtmbp is therefore a result of the closed conformation in isolation, while the reduced ATPase observed in DDM likewise results from the inability of MBP-C to open. Design of a Stabilized-Open Maltose Binding Protein Having seen that the closed conformation of MBP in isolation displays a markedly different ability to stimulate the transporter from the wild type protein, 136

148 Fig 4.5 Changes in SAXS Profile of MBP-C with Maltose SAXS intensity curves are shown for MBP-wt and MBP-C overlaying the curves of the proteins with and without maltose (above). The difference curves of these profiles are shown, indicating the degree of change on the addition of maltose to wtmbp and MBP-C (below). 137

149 138

150 Table 4.2 Radii of Gyration of MBP-C from GNOM Analysis Protein Ligand Radius of Gyration (Å) wtmbp ± 0.02 wtmbp 5mM Maltose 21.8 ± 0.03 MBPC ± 0.04 MBPC 5mM Maltose 21.9 ±

151 the investigation was continued by attempting to isolate and stabilize the open form of MBP. The tendency of MBP to adopt a closed conformation is normally resisted, and largely prevented in the absence of maltose, by a region of MBP known as the balancing interface, which comprises a series of hydrophobic contacts between the two domains [11]. When MBP binds maltose these contacts are abolished by domain rotations, such that their formation influences the equilibrium between open and closed MBP (Fig. 4.6A). Disrupting favorable interactions in the balancing interface is known to shift MBP towards a closed conformation [11]. It was theorized that stabilization of the balancing interface would disfavor the closed conformation, potentially holding MBP in the open state. An open stabilized MBP would enable examination of the interactions between the open conformation and the transporter without the complicating influence of a conformational equilibrium between open to closed MBP. We used the available X-ray crystal structures of MBP [4,5,8] to incorporate a potential disulfide bridge that spans the balancing interface to link the N- and C- terminal domains. Cysteines were introduced at positions G101C and G174C (Fig. 4.6A). Neither position is predicted to make contacts with the transporter. These positions were selected such that a disulfide bond between them would be compatible with the open conformation seen in the transporter ATPase catalytic state but not with the closed conformation. The sulfur atoms of the mutated residues are 4.0 Å apart in the open form of the protein but are separated by 14.8 Å in the closed maltose bound state. A disulfide bond between them should therefore 140

152 Fig 4.6 MBP-O Cysteine Mutations Cysteine mutations (yellow) are shown mapped to X-ray crystal structures of open and closed MBP. The maltose-binding site is shown as a yellow wedge, with bound maltose (green and red). The hydrophobic balancing interface is highlighted in blue. (A.) MBP-O mutations G101C and G174C are shown. (B.) MBP-O95/171 mutations D95C and Y171C are shown. PDB Reference 2R6G [8], 1ANF [4] 141

153 142

154 prevent the protein from adopting a closed conformation by destabilizing that state through bond length constraints. This mutant binding protein is referred to as MBPopen (MBP-O) in reference to its predicted favoring of the open state. When MBP-O was purified it proved resistant to direct oxidation by CuCl2. We instead chose to cross-link the mutant by modification with the bi-functional cysteine-reactive linker Bis(maleimido)ethane (BMOE). BMOE has an effective length of 8.0 Å which could allow MBP-O greater conformational flexibility than a direct disulfide bond between residues 101 and 174. We therefore designed and purified an alternative mutant with mutations D95C and Y171C. This mutant, referred to as MBP-O95/171 has more widely spaced cysteines in the open state and should therefore be more constrained by BMOE (Fig. 4.6B). These mutations were also not predicted to contact the transporter. When purified MBP-O and MBP-O95/171 were reacted with BMOE, samples were taken of the reaction at successive time points and reacted with excess fluorescene maleimide. SDS-PAGE and fluorescence imaging revealed that the reaction reduced the availability of free cysteines to be labeled, with no detectable labeling in the final preparation (Fig. 4.7). This indicated that all cysteine residues were modified by BMOE. The BMOE-modified MBP-O and MBP-O95/171 were next analyzed by Matrix Assisted Time of Flight (MALDI-TOF) mass spectrometry to confirm its modification (Table 4.3). The molecular masses of both proteins indicated the covalent attachment of a single BMOE molecule. Further, examination of the mass spectra of each protein indicated a single homogenous peak, without detectable contamination from unmodified protein species, or protein cross-linked 143

155 Fig 4.7 Fluorescene Labeling of BMOE Cross-Linked MBP-O. BMOE was repeatedly added to reduced MBP-O in a 1:2 molar ratio at 30 minute time intervals. Samples were removed at 30 minute intervals, arrested with excess Fluorescene maleimide (FM) and fluorescence was measured after SDS- PAGE. Lane 1: Reduced MBP-O fully labeled with FM, Lanes 2-10: time course of cross linking reaction (minutes), Lane 11: Over-night incubation of reaction and final product. 144

156 145

157 Table 4.3 MALDI-TOF Analysis of Prepared Binding Proteins MBP mutant Modification Predicted Mass(Da) Observed wtmbp No mod MBP-O No mod MBP-O 2 BMOE MBP-O* 1 BMOE MBP-O95/171 No mod MBP-O95/171* 1 BMOE *Protein sample used for further studies 146

158 Fig 4.8 MALDI-TOF Profile of Cross-Linked MBP-O and MBP-O95/171. Mass spectra were collected of the purified and crosslinked MBP-O and MBP- O95/171 binding proteins in Linear Mode on a MALDI-TOF mass spectrometer. The dominant peaks are circled for MBP-O and MBP-O95/

159 148

160 to two BMOE molecules (Fig. 4.8). With both mutants demonstrating the blocking of two cysteines by a single molecule of BMOE it was concluded that the protein had been correctly modified, with a single BMOE molecule spanning two cysteines across the balancing interface. Stimulus of the wild-type maltose transporter by BMOE modified mutants Surprisingly, when MBP-O was added to PLS containing MalFGK2 a large degree of ATPase activation was seen, comparable to full activation by MBP-maltose (Fig. 4.9A). The addition of maltose did not significantly change this activation, even at 10 mm. This activity was also seen with MBP-O95/171, indicating that the effect is not a unique feature of the mutations in MBP-O (Fig. 4.9B). This high degree of activation by both MBP-O mutants was highly unexpected, as the conformation of MBP-O is predicted to closely mimic that of wtmbp, which activates very little ATPase in the absence of maltose. In both mutants a BMOE cross-link across the MBP balancing interface has induced a high level of uncoupled ATPase in the absence of maltose, although the mechanism underlying this effect is unclear. Both MBP-O mutants were also added to detergent solubilized MalFGK2 to test whether the maltose indifferent phenotype would be retained. In the DDM solubilized system the addition of MBP-O and MBP-O95/171 stimulates high rates of ATP hydrolysis, comparable to that of wtmbp and wtmbp-maltose (Fig. 4.10AB). MBP-O activates a high level of ATP hydrolysis that is not affected the presence of maltose (Fig. 4.10A). Whereas MBP-O95/171 stimulates high activity at lower concentrations (2-50µM), activation appears to drop as the transporter becomes 149

161 Fig 4.9 ATPase Stimulus by MBP-O in Proteoliposomes. Purified MalFGK2 was reconstituted into Proteoliposomes and incubated at 37 C with (A.) MBP-O, and MBP-O with maltose and (B.) MBP-O95/171. The rate of ATP hydrolysis was calculated as described in Fig

162 151

163 Fig 4.10 ATPase Stimulus by MBP-O of Detergent Stabilized MalFGK2. Purified MalFGK2 was extracted from the cell membrane and solubilized in 50 mm Tris ph7, 4mM MgCl2, 5% glycerol, 0.01% dodecyl-maltoside [15]. The resulting uncoupled transporter was incubated at 37 C with A. wtmbp, wtmbp with maltose(5mm) and B. MBP-O95/171. The resulting rate of ATP hydrolysis was calculated as described in Fig

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165 saturated with binding protein. This difference from wtmbp and MBP-O may be a result of reduced conformational flexibility in MBP-O95/171. Characterization of MBP-O mutants The high levels of constitutive ATPase activity seen on the addition of either MBP-O mutant to wild type MalFGK2, were very unusual, so the conformational mechanics of the mutants were investigated to better interpret their phenotype. Modified MBP-O and MBP-O95/171 were both retained on the resin during amylose chromatography, which indicated maltose binding. Analysis of intrinsic tryptophan fluorescence quenching showed that MBP-O has a wild-type affinity for maltose, with a KD (concentration at half maximal binding) of 4.5 ±0.4 µm, nearly identical to the wild-type KD of 4.4 ±0.5 µm (Fig. 4.4). These results indicate that BMOE modification was not sufficient to abrogate maltose binding. SAXS analysis was performed on both mutants to observe conformational changes in the proteins on maltose binding. The program GNOM was used to derive the radius of gyration (Rg) of the proteins. In the relaxed state without maltose, MBP-O displays an Rg of 23.0 Å, similar to the 22.8 Å Rg of wtmbp. MBP-O95/171 adopts a more closed conformation, with an Rg of 22.5 Å (Table 4.4). To test whether this change was a result of the crosslink a sample of MBP-O95/171 was modified with N-ethylmaleimide (NEM) rather than BMOE. NEM covalently blocks each cysteine similar to BMOE modification, but each cysteine is modified by a separate molecule of NEM, so the two sites are not cross-linked in a way that constrains conformational changes. MBP-O95/171 modified in this way 154

166 Table 4.4 Radii of Gyration of MBP-O and MBP-O95/171 from GNOM Analysis Protein Modification Ligand wtmbp - - wtmbp - 5mM Maltose MBPO BMOE - MBPO BMOE 5mM Maltose MBPO95/171 BMOE - MBPO95/171 MBPO95/171 MBPO95/171 BMOE NEM NEM * Average of 4 measurements ** Average of 5 measurements 10mM Maltose 5mM Maltose Radius of Gyration (Å) 22.8 ± ± * 22.3 ** 22.5 ± ± ± ±

167 demonstrated a more open conformation with an Rg of 23.3, indicating that the reduced Rg seen with BMOE modified MBP-O95/171 is a result of the crosslink between mutant cysteines (Table 4.4). On the addition of maltose both mutant proteins show a reduction in Rg that indicates a conformational change towards a more compact, and likely more closed, structure. Both mutants display a smaller reduction in Rg than wtmbp. However, because MBP-O95/171 is more compact in the relaxed state this change results in a more compact maltose-bound conformation than wtmbp. The NEM modified sample of MBP-O95/171 demonstrated a reduction in Rg similar to wtmbp (Table 4.4). These data indicate that BMOE modification has not constrained the mutant proteins to an open conformation or prevented maltose binding. These findings are corroborated by a broader analysis of the full SAXS spectra of the mutants. A substantial shift is seen in the scattering spectra of each protein with and without maltose, as indicated by difference curves of those spectra (Figure 4.11). BMOE modified mutants however, display a lesser shift, which suggests some constraint on conformational changes. This analysis indicates that while both mutant proteins are modified with BMOE, they are still able to bind maltose and are not restricted solely to the open conformation. However, despite the modest observable influence BMOE has on MBP conformational changes, it has resulted in rapid constitutive ATPase activation of MalFGK2. 156

168 Fig 4.11 Changes in SAXS Profile of MBP-O and MBP-O95/171 with Maltose SAXS intensity curves are shown for MBP-wt, MBP-O, MBP-O95/171 and MBP-O95/171 modified instead with NEM. The curves of the proteins with and without maltose are overlaid (above). The difference curves of these profiles are shown, indicating the degree of change on the addition of maltose to each protein (below). 157

169 158

170 4.4 Discussion In this study I have observed that each of three conformationally stabilized MBP mutants, MBP-C, MBP-O and MBP-O95/171, are able to activate significant amounts of ATPase activity. Previous studies have demonstrated that both the open and closed forms of MBP interact with different conformations of the transporter [8,9,10,27]. Here it has been shown that both of these interactions promote ATP hydrolysis. MBP-C adopts the closed conformation, even in a solution lacking maltose. The dominant negative phenotype observed by Zhang et al demonstrates that MBP- C does not open sufficiently to release maltose into the transporter, and therefore cannot promote transport [16]. This indicates that the protein is constrained to the closed state even when bound to MalFGK2. Here we show that despite this limitation, the protein is significantly activating for ATPase. Concerns that the observed activation in proteolipsomes might be caused by undetected contamination from reduced MBP-C are refuted by the high level of activation observed. Further, the rate of ATPase is far in excess of the maximal rate stimulated by excess unliganded wtmbp. This activity is also not increased by the addition of maltose, unlike reduced MBP-C or wtmbp, which is not consistent with contamination. The ability of MBP-C to activate the wild type transporter indicates that the closed form in isolation is sufficient to stimulate some ATPase activity. However activation by MBP-C displays a defect, as the rate of hydrolysis supported by MBP-C is considerably less than the maximal rate activated by wtmbp with maltose. 159

171 Previous work in this thesis has shown that the substrate does not play a direct role in ATPase activation, so the defect must arise from the inability of the binding protein to adopt the open conformation [17]. This is consistent with a model of maltose transport in which MBP domain opening is a significant step in the ATPase catalytic cycle. However, the large degree of stimulus by MBP-C, relative to the basal rate, indicates that the closed conformation of MBP is sufficient to partially complement the need for MBP. The closed conformation of the protein must therefore trigger the complex to progress into the catalytic cycle. Note that these results complement previous findings in chapter 2 of this thesis that transient, or unstable, closed species of MBP do not activate ATPase [12:chapter2]. In that work, MBP was forced towards the closed conformation by mutations or deletions within the balancing interface. These mutations disfavored the open conformation of MBP but did not stabilize the closed state, whereas the closed form of MBP-C is highly stabilized by both a disulfide bond and captive molecule of maltose. The resulting mutants showed reduced ATPase stimulation in the presence of maltose, displaying a defect in activation consistent with our findings for MBP-C. However unlike MBP-C, these mutants did not become more activating for ATPase in the absence of maltose. Further, it was found that the ATPase stimulus observed from unliganded MBP-wt was not consistent with causation by transiently closed species of MBP. Taken together these results indicate that the closed conformation of MBP is only activating for ATPase when it is stabilized and energetically favorable and that these features must play a role in its mechanism of activation. 160

172 These results are consistent with a model of activity by which closed MBP selects for an otherwise transient conformation of the transporter by binding and stabilizing a high-energy intermediate. The closed form of MBP could mediate access to the ATPase catalytic state of the transporter by binding and stabilizing the pre-catalytic state; flattening or lowering an unfavorable energetic barrier in the same way that enzyme accelerated reactions lower the activation energy by stabilizing disfavored chemical transition states [28,29]. Indeed, it is the precatalytic state of the transporter, intermediate between the cytoplasmic facing resting state and periplasmic facing catalytic state, which has been observed through X-ray crystallography and paramagnetic resonance imaging to bind closed MBP [9,30]. When MalFGK2 was solubilized by a mild detergent, ATP hydrolysis was activated at a high rate by wtmbp, wtmbp with maltose, MBP-O and MBP-O95/171; all of the binding proteins capable of adopting the open conformation. MBP-C however, being unable to adopt the open state, stimulated significantly less ATPase. This result is particularly striking when contrasted with the behavior of unliganded wtmbp. ATPase activation by the two proteins is differently impacted by detergent solubilization of the transporter. While the ability of MBP-C to activate the transporter is reduced relative to background, wtmbp activates a far greater increase in ATPase in detergent than in proteoliposomes. This is strong evidence that the two proteins are activating the transporter through different mechanisms; targeting distinct conformations of the transporter which are differently disrupted by detergent solubilization. 161

173 In previous work it was shown that unliganded wtmbp stimulates ATPase by targeting the small fraction of transporter able to transiently adopt the catalytic state [12]. The crystal structure of the catalytic state transporter shows that the catalytic state is the conformation of MalFGK2 bound by unliganded MBP [8]. That detergent solubilization increases the ATPase stimulus of unliganded wtmbp suggests that the catalytic state of the transporter has become more accessible to MBP. Note that this increased availability of catalytic state MalFGK2 is correlated with a decrease in the ability of MBP-C to activate the transporter above background. These results are consistent with a role for closed MBP in binding and stabilizing the pre-catalytic state to promote the appearance of catalytic state MalFGK2. Detergent solubilization has removed the transporter from its native membrane environment and altered the relative energies and frequencies of its competing conformations. The increase in background ATPase suggests that this alteration has made the catalytic state more accessible. Doing so has made wtmbp, which directly targets the catalytic state, more activating and simultaneously renders the ability of MBP-C to stabilize the pre-catalytic state less significant. Detergent solubilization has already lowered the energy of activation of the catalytic cycle by stabilizing the pre-catalytic state. This likely explains the ability of Oldham and Chen to crystallize the pre-catalytic state transporter in detergent [9]. The behavior of both MBP-O and MBP-O95/171 in liposomes was a great surprise. The mutations and BMOE modification we introduced into MBP-O and MBP-O95/171 had been intended to hold those proteins in the open conformation. Modification proved insufficient to reduce the proteins maltose-binding affinity, but 162

174 SAXS analysis indicated that the ability of each mutant to close had been altered. A profound effect on activity was observed however, as both BMOE modified mutants stimulate high levels of ATPase from proteoliposome embedded MalFGK2 without the addition of maltose. This represents a pronounced gain-in-function in the transporter brought on solely by changes in MBP; with constitutive ATPase freed from coupling to maltose transport. A change in the relative favorability of the open and closed conformations should have revealed itself as a change in the binding affinity of the protein for maltose as has been previously observed in other mutants [12]. However, as both SAXS and intrinsic fluorescence quenching experiments observe the protein at an equilibrium state, it is possible for changes in the kinetics of the protein to go undetected. During the ATPase catalytic cycle the binding protein undergoes significant changes in conformation and dynamically alters its interaction with the transporter. A change in the rate of conformational change would significantly impact the ATPase catalytic cycle even if it did not alter substrate binding behavior at equilibrium. This is likely what has occurred in MBP-O and MBP-O95/171 stimulated ATPase trials. Despite an ability to bind and close around maltose, MBP-O stimulates a high level of activity in either the presence or absence of substrate. That there is little or no change in the stimulated rate of hydrolysis between liganded and unliganded forms of MBP-O suggests that the closed conformation is not playing a significant role in the uncoupled activity observed. This is an indication that the normal catalytic cycle has been short-circuited. Although comparatively little is known 163

175 about the dissociation mechanics of the transporter following ATP hydrolysis, conversion of the catalytic state, which binds open MBP with high affinity, to the resting state which does not, will necessarily involve a loss of affinity for open conformation MBP. Paramagnetic resonance studies of the post-catalytic transporter seem to indicate that it reverts to the resting state via rigid body rotations which are broadly the reverse of changes prior to catalysis [1]. This means that the interface between the catalytic state transporter and open MBP will move towards a conformation that is more consistent with closed conformation MBP, likely causing wtmbp to dissociate as the closed state is energetically disfavored. In the case of MBP-O and MBP-O95/171, the BMOE modification might delay or resist conformational changes in the binding protein. This could cause the transporter to become arrested in a catalytic-like conformation long enough for ADP in the ABC subunits to be exchanged for ATP, priming the transporter for repeat hydrolysis. This is supported by the high affinity of open MBP for the catalytic state transporter, as observed by its failure to dissociate during size exclusion chromatography [27]. The high affinity of open MBP for the transporter suggests it would have significant binding energy with which to influence the conformation of the complex before being forced to dissociate. This mechanism could be very similar to that by which maltose transport is inhibited by wtmbp in MBPindependent transporter mutants, which have mutationally stabilized catalytic states [25,31,32]. In each case, a change in the conformational dynamics of 164

176 competing MBP and transporter conformations disrupts the normal ATPase catalytic cycle and prevents the complex from dissociating. It is possible that the mutations or BMOE linker incorporated into MBP-O and MBP-O95/171 have disrupted some ATPase relevant interaction between local residues of MBP and the transporter. However this is unlikely, as all mutations are near the MBP balancing interface, which is not on the juxtamembrane facing of MBP. The P2 arm of MalF intrudes into the periplasm to contact portions of MBP that are not on the juxtamembrane surface and this arm has been implicated in conformational coupling between MBP and MalFGK2 [33,34,35]. However, the MBP- O mutants used here were designed with this risk in mind and consequentially the introduced cysteines are not proximal to any sites of contact between MBP and the transporter. Further, as both MBP-O and MBP-O95/171 displayed similar uncoupled ATPase phenotypes, despite their lack of common mutations, it is less likely that the introduced cysteines account for this phenotype directly. In the future, X-ray crystal structures of the BMOE cross-linked mutants will help to interpret what effect the BMOE modification has had on the MBP conformational equilibrium. 165

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180 Chapter 5 Discussion and Conclusions At t h e co m m en cem en t o f t h i s t h es i s i t was b el i ev ed t h at o n l y t h e cl o s ed co n fo rm at i o n o f M BP p l a ye d a s i gn i fi c an t ro l e i n ATP h yd ro l ys i s b y t h e m al t o s e t ran s p o rt er. Th i s w as l ar gel y d u e t o t h e ab i l i t y of m al t o s e t o i n d u ce M BP d o m ai n cl o s u re [ 1,2,3,4,5 ]. Du ri n g t h e wo rk o f t h i s t h es i s a g ro un d b reak i n g M al FGK 2 cr ys t al s t ru ct u re was res o l v ed b y an o t h er gro up, s h o wi n g t h e i n t e ra ct i o n o f op en M BP wi t h t h e ATP as e cat al yt i c co n fo rm at i o n o f t h e t ran s p o rt er, wh i ch fa ces t h e p e ri p l as m [ 6 ]. La t er, a r es t i n g- s t at e s t ru ct u re w as res o l v ed wh i ch s h o wed t h e t ran sp o rt er i n a c yt op l as m i c f aci n g, an d wi t h o u t a b i n d i n g s i t e fo r M BP [ 7 ]. Fi n al l y, a p re - cat al yt i c s t at e s t ru ct u r e i l l u s t rat ed t h e i n t eract i o n o f cl o s ed fo r m M BP wi t h a t ran s p o rt e r co n fo rm at i o n i n t erm ed i at e b et we en t h e res t i n g an d cat al yt i c s t at e s [ 8 ]. Th es e s t ru ct u res ar e t rem en d o u s co n t ri b u t i o n s t o t h e u n d ers t an d i n g o f M al FG K 2 fu n ct i o n an d a gre e wi t h t h e res u l t s o f p rev i o u s i n-s o l u t i o n s t u d i es o f t ran s p o rt er co n fo rm at i o n [ 9,1 0,1 1,1 2 ]. Th es e fi n d i n gs h el p ed t o d efi n e t h e co n fo rm at i o n al ch an ges u n d ert ak en d u ri n g m al t o s e t ran s p o rt an d t h e i n t e ra ct i o n s b et ween t h e b i n d i n g p ro t ei n an d t r an s p o rt er. Ho wev e r t h e f u n ct i o n al s i gn i fi c an ce o f t h es e i n t eract i o n s was n o t u n d e rs t o o d. Fu rt h er t h e m ech an i s m b y wh i ch t h es e i n t eract i o n s rev ers e t h e au t o i n h i b i t i o n o f ATP as e an d i n s t e ad a ct i v at e t h e t ran s p o rt er w a s n o t u n d ers t o o d, as i n d i cat ed b y t h e l o ck -an d -k e y h yp o t hes i s, wh i ch res t ed o n a t aci t as s u m p t i o n t h at u n l i gan d ed cl o s ed M BP wo u l d ac t i v ate t h e t ran s p o rt e r t h ro u gh s i m p l e s u rfac e co m p l em en t a ri t y [ 1 3,3 ]. Th e p u rp o s e o f t h e res ear ch p res en t ed i n t h i s t h esi s was t o i d en t i f y t h e m ec h an i s m b y wh i ch ATP h yd ro l ys i s i n t h e m al t o s e t ran s p o rt er i s re gu l at ed an d co up l ed t o s ub s t rat e t ran s p o rt ; h o w t h e t ran s m em b ran e (TM ) s ub u n i t s o f t h e co m p l ex can i n h i b i t ATP 169

181 h yd ro l ys i s i n t h e res t i n g s t at e, b u t act i v at e ATP as e i n res p o n s e t o m al t o s e b o u n d M BP. Th i s m ech an i s m h as i m p l i cat i o n s fo r o u r u n d ers t a n d in g o f t r an s p o rt ers acr o s s t h e wh o l e ABC fam i l y. B as ed o n t h e fi n d i n gs p res en t ed h er e, a m o d el o f t r an s p o rt er re gu l at i o n h as b een d ev el op ed t o ex p l ai n t h e ob s erv ed b eh av i o r ( Fig 1.4 ). In t h i s m o d el, u n co up l ed ATP h yd ro l ys i s i s i n h i b i t ed b y en er g et i cal l y d i s fa vo ri n g co n fo rm at i o n s o f t h e t ran s p o rt er t h at are co m p et en t fo r A TP as e. W h i l e i n i s o l at i on t h e M al K d i m er c an fr e el y cl o s e an d h yd ro l yz e ATP, wh en i n co rp o rat ed i n t o t h e fu l l M al FGK 2 co m p l ex t h i s m o t i o n b eco m es co up l ed t o h i gh l y u n f av o rab l e co n fo rm at i o n al ch a n ges wi t h i n t h e M al FG T M re gi o n s. Th e res t i n g s t at e co n fo r m at i o n t h erefo r e rep res e n t s a v al l e y i n t h e en e r ge t i c l an d s cap e o f t h e co m p l ex wh i ch i s b o u n d ed b y d i s f av o r ed, h i g h -ener g y, al t ern at i v e co n fo rm at i o n s. Th i s fo rm s t h e fi rs t en e r get i c b arri er b y wh i ch th e t ran s p o rt e r i s eff ect i v el y r es t ri ct ed t o co n fo rm at i o n s n ea r i t s c yt op l as m -faci n g r es t i n g s t at e, wh i ch i s i n act i v e wi t h res p ect t o ATP h yd ro l ys i s ( Fi g 1.4 ). Ho wev e r, wh en t h e cl o s e d m al t o s e-b o u n d fo rm o f M BP t ar get s an d b i n d s t h e p recat al yt i c s t at e o f t h e t ran s p o rt er i t l en d s s o m e o f i t s co n s i d erab l e co n fo rm at i o n al s t ab i l i t y t o t h e co m p l ex. Th i s h as t h e eff ect o f s el ect i n g, a n d fav o ri n g, t h e p r e-c at al yt i c s t at e o u t o f an eq u i l i b ri u m t h at i s n o rm al l y b i as ed o v erwh e l m i n gl y t o w ard s t h e r es t i n g s t at e. Th e h i gh en er get i c b arri e r rep res en t ed b y t h e p re- cat al yt i c s t at e i s t h er efo r e l o w ered an d t h e en er get i c l an d s cap e fl at t e n ed, wh i ch i n cr eas es t h e t en d en c y o f t h e t ran s p o rt er t o res i d e i n co n fo rm at i o n s o t h er t h an t h e res t i n g s t at e. In t h is wa y t h e c at al yt i c s t at e o f t h e t ran s p o rt er i s m ad e m o re acc es s i b l e b y red u ci n g t h e act i v at i o n en e r g y n e ces s ar y t o ex i t t h e res t i n g s t at e. 170

182 Th e wo rk p r es en t ed h ere ag rees wi t h an d s up p o rt s t hi s m o d el, s u gg es t i n g t h at t h e ro l e o f cl o s ed M BP i s t o m ed i at e t h e co n v e rs i o n o f t h e res t i n g s t at e t ran s p o rt er t o t h e ATP as e cat al yt i c s t at e b y s t ab i l i z i n g t h e p re-c at al yt i c s t at e. Th i s can b e s ee n i n t h e cap a ci t y o f M BP -C an d s M BP wi t h s u cro s e t o a ct i v at e h yd ro l ys i s, wh i l e u n l i gan d ed cl o s ed -wt M BP, M BP -D M, an d M BP A9 6 W / I3 2 9 W are al l i n cap ab l e o f s t i m u l at i n g h i gh l ev el s o f act i v i t y. Al l t h es e b i n d i n g p ro t ei n s a re ca p ab l e o f ad op t i n g t h e cl o s ed co n fo rm at i o n, h o wev er o n l y t h e p ro t ei n s wh i ch ar e en er g et i cal l y s t ab l e i n t h at s t at e ar e cap ab l e o f act i v at i n g t h e t ran s p o rt er. O f p art i cu l ar n o t e i s t h e co n t ras t o f M BP A9 6 W / I3 2 9 W wi t h M BP -C. Nei t h er o f t h es e p ro t ei ns ad op t s t h e op e n co n fo rm at i o n, an d b o t h a re h el d n e ar t o t h e cl o s ed s t at e. Ho wev er M BP -C, wh i ch i s s t ab i l i z ed b y a d i s u l fi d e b o n d, i s s i gn i fi can t l y act i v at i n g fo r ATP as e wh i l e M BP A9 6 W / I3 2 9 W, wh i ch h as an av er a ge co n fo r m at i o n v er y n ea r t h e cl o s ed s t at e b u t i s n o t en er get i c al l y f av o r ed i n t h e fu l l y cl o s ed co n fo rm at i o n, i s wh o l l y i n cap a b l e o f act i v at i n g h yd ro l ys i s wi t h o u t b ei n g s t ab i l i z ed b y m al t o s e. W h en M BP A9 6 W / I3 2 9 W i s s t ab i l i z ed i n t h e cl o s ed s tat e b y m al t o s e, b u t s t i ll in cap ab l e o f ad op t i n g a fu l l y op en s t at e, i t ap p ears t o s t i m u l at e ~2 0 % o f t h e m ax i m al act i v i t y o f wt M BP -m al t o s e ( Fi g 2.8 an d 4.3 ). Th i s i s a rat e o f h yd ro l ys i s s i m i l ar t o t h at s e en wi t h M BP -C, b ro u gh t o n b y s t ab i l i z at i o n o f t h e cl o s ed fo rm o f M BP A9 6 W / I3 2 9 W. Ad d i t i o n al l y, i n ch ap t er 2 i t was fo u n d t h at u n l i gan d ed cl o s ed -M BP, as op p o s ed t o u n l i gan d ed op en -M BP, was n o t cap ab l e act i v at i n g t h e t ra n s p o rt er, d es p i t e ev i d en c e t h at s u ch a s p e ci es ex i s t s i n s o l u t i o n [ 1 4 ]. Th es e res u l t s d em o n s t rat e t h at t h e ab i l i t y o f cl o s ed M BP t o act i v at e t h e t ran s p o rt e r i s a fu n ct i o n o f i t s co n fo r m at i o n al s t ab i l i t y, o r p ers i s t en ce, as t h o s e b i n d i n g p ro t ei n s 171

183 wh i ch ad op t o n l y a t ran s i en t l y cl o s ed co n fo rm at i o n h av e n o en er g y b u d g et i n b i n d i n g t o t h e p re- cat al yt i c s t at e t r a n s p o rt er wi t h wh i ch t o co n t ri b u t e t o t h e co m p l ex es s t ab i l i t y. Ho wev e r, t h i s i n t eract i o n i s n o t s u ffi ci en t fo r ful l t ran s p o rt er act i v i t y. Th e M al K ABC d i m er i s o n l y p art i a l l y cl o s ed i n t h e p r e-c at al yt i c s t at e, m ak i n g i t u n l i k el y t h at t h i s co n fo rm at i o n c an h yd ro l yz e ATP at a s i gn i fi c an t rate [ 8 ]. Fu rt h er, at t h i s p o i n t i n t h e cat al yt i c c ycl e m al t o s e i s s t i l l en cl o s ed wi t h i n M BP o n t h e p eri p l as m i c m e m b ran e f ace. To fu l fi l l i t s p h ys i o l o gi ca l ro l e t h e t ran s p o rt e r m u s t en s u re t h at m al t o s e en t e rs t h e t ran s l o cat i o n p at h wa y b e fo re t h e co m p l ex rev ert s t o i t s res t i n g s t at e. To t h i s en d, i t i s l i k el y t h at t h e en er g y o f ATP b i n d i n g i s real i z ed a t t h i s s t age, b y an en er g et i c cl o s u re o f t h e M al K s ub u n i t s aro u n d t h ei r b o u n d ATP m o l ec u l es. Di m er cl o s u re b eco m es fav o rab l e wh en t h e t ran s p o rt er en t e rs t h e p re- cat al yt i c s t at e. Th i s co u l d b e a res u l t o f M BP l o weri n g t h e en t rop y o f t h e res t i n g s t at e t ran s p o rter an d s o r ed u ci n g t h e en t rop y co s t o f d i m er cl o s u re. A BC d i m er cl o s u re wi l l m o v e t h e t ran s p o rt er t o w ard s t h e A TP as e cat al yt i c co n fo rm at i o n (F i g 1.4 )[ 6 ]. Th e m o t i o n o f M al K d i m er cl o s u re i s co m m u n i cat ed acro s s t h e m em b ran e t h ro u gh ri gi d b o d y ro t at i o n s o f t h e TM s ub u n i t s an d fo rces M BP t o op en [ 7 ]. Wi t h M BP b o u n d i n t h e op en s t at e, m al t o s e i s ex p o s ed t o t h e t ran s l o cat i o n p at h wa y, a n d i t s affi n i t y fo r M BP h as b een r ed u ce d, o wi n g t o t h e op en i n g o f t h e M BP m al t o s e b i n d i n g s i t e. Ho wev e r, t o effi ci en t l y c o up l e ATP h yd ro l ys i s t o t ran s p o rt, ATP as e m u s t b e d el a yed u n t i l t h e s ub s t rat e h as b e en reco gn i z ed as l eav i n g M BP an d en t e red t h e TM re gi o n s. Th i s i s l i k el y ac co m p l i s h ed b y t h e M al G P 3 s co op l o o p [ 6 ]. Th e s co op l o op i nv ad es t h e M BP m al t o s e-b i n d i n g s i t e o n ce m al t o s e h as en t er e d th e TM re gi o n s. It i s t h erefo re a s en s o r wh i ch c an d et e rm i n e t h e p o s i t i o n o f m al t o s e wi t h o u t d i rect l y i n t e rac t i n g wi t h t h e 172

184 s ub s t rat e. Th e l o op i s s t ru ct u ral l y l i n k ed t o t h e C - t erm i n al t ai l o f M al G, w h i ch i s p art o f an ex t en s i v e h yd ro g en b o n d n et wo rk i n t h e cen t er o f t h e M al K d i m er. Th i s s t ru ct u re fo rm s a l i k el y t ran s m i s s i o n m ech an i s m t o l i n k ATP h yd ro l ys i s t o t h e ab s en ce o f m al t o s e wi t h i n t h e M BP m al t o s e b i n d i n g s i t e. W h i l e i t was p rev i o u s l y r eco gn i z ed t h at op en M B P b in d s wi t h h i gh affi n i t y t o t h e cat al yt i c s t at e co m p l ex t h ere w as n o fu n ct i o n al ev i d en ce t h at t h i s i n t era ct i o n p ro m o t es ATP h yd ro l ys i s. Th i s t h es i s h as s h o wn t h at t h e o p en co n fo rm at i o n o f M B P i s cap ab l e o f act i v at i n g co n s i d er ab l e A TP h yd ro l ys i s d i s t i n ct an d s ep arat e fro m a ct i v at i o n b y cl o s ed M BP. In ch ap t er 2 i t wa s fo u n d t h at op en M BP, r at her t h an cl o s ed, was r e s p o n s i b l e fo r t h e ATP as e a ct i v at i o n s t i m u l at ed b y u n l i gan d ed M BP. In ch ap t er 4 t h i s was d ev el op e d fu rt h er i n ex p eri m en t s wi t h t h e d et er gen t s o l ub i l i zed t ran s p o rt e r wh i ch s h o wed t h at b o t h M BP -C an d u n l i g an d ed wt M BP s t i m u l at ed ATP as e act i v i t y, b u t d o s o b y m ech an i s m s wh i ch are d i ffe ren t l y i m p act ed b y d et er gen t s o l ub i li z at i o n. Th i s d i fferen t i al effe ct i s a s t ro n g i n d i cat i o n t h at t h e op en an d cl o s ed fo rm s o f M BP s t i m u l at e t h e t ran s p o rt er b y t arg et i n g d i ffe ren t co n fo r m at i o n s o f M al FGK 2. A p rev i o u s s t u d y u s i n g a m u t an t M al FGK 2 co m p l ex i s al s o co n s i s t en t wi t h t h i s fi nd i n g, as i t was fo u n d t h at a m u t an t, wh i ch was p r es u m ab l y a b l e t o ad op t t h e cat al yt i c con fo rm at i o n d u e t o a l o wered fi rs t en er get i c b arri er, w as act i v at ed b y op en wt M BP t o tran s p o rt a s ub s t rat e t h at d o es n o t i n d u ce M BP d o m ai n cl o s u re [ 1 5 ]. Fi n al l y t h e h i g h rat es o f ATP h yd ro l ys i s s een fro m BM OE m o d i fi ed M BP -O an d M BP -O9 5 / 1 7 1, wh i ch wer e i n d i ffe ren t t o t h e p res en c e o r ab s en ce o f m al t o s e, a re s t ro n g ev i d en ce t h at t h e b in d i n g o f op en M BP t o t h e cat al yt i c s t at e i s a fu n ct i o n al i n t er act i o n b y wh i ch M BP p r o m o t es ATP h yd ro l ys i s. Th e fu n ct i o n al s i gn i fi can ce o f t h e M al G s co op l o op was re co gn i z ed i n ch ap t er 3. Th e eq u i v al en t rat es o f 173

185 ATP h yd ro l ys i s s t i m u l at ed b y s M BP wi t h m al t o s e o r s u cro s e s h o w ed t h at A TP h yd ro l ys i s i s n o t i n fl u en c ed b y d i r ect i n t er act i o n s b et ween t h e s ub s t r at e an d t h e TM d o m ai n s. Th e red u c ed r a t e o f h yd ro l ys i s s t i m u l at ed b y t h e m u t an t, as co m p ared t o wt M BP, i n d i cat ed t h at i n t eract i o n s b et w een t h e m al to s e b i n d i n g s i t e an d s c o op l o op p ro m o t e ATP as e ( Fi g ). Th i s red u c ed act i v i t y was s een ev en wh en s M BP was s t ab i l i z ed i n t h e cl o s ed s tat e b y m al t o s e o r s u c ro se. Th es e res u l t s i n d i cat e t h at t h e op en fo rm o f M BP p l a ys a m aj o r fu n ct i o n al ro l e b y al l o win g t h e cat al yt i c s t at e t r an s p o rt er t o o v erco m e a s eco n d en er g et i c b arri er b y wh i ch A T P h yd ro l ys i s i s o t h erwi s e p rev en t ed (Fi g. 1.4 ). It s h o u l d b e n o t ed t h at w h i l e u n l i gan d ed op en wt M BP was s een t o act i v at e ATP as e i n p ro t eo l i p o s o m es [ 1 3 ], an d wh i l e t h i s i s hi gh l y rev eal i n g, t h i s act i v i t y i s n o t l i k el y s i gn i fi can t in vivo. Th e cat al yt i c t r an s p o rt er co n fo rm at i o n t ar g et ed b y op en M BP i s effect i v el y d o wn -s t re a m o f act i v at i o n b y cl o s ed M BP, l i m i t i n g t h e rat e o f u n co up l ed h yd ro l ys i s i n t h e ab s en ce o f m al t o s e. Ho wev er, as d et er gen t s o l ub i l i z at i o n s t u d i es h av e s h o wn, rem o v i n g t h e t ran s p o rt er fro m i t s n at i v e en vi ro n m en t rel ax es i t s au t o i n h i b i t i o n o f ATP as e b y l o weri n g t h e fi rs t en er g et i c b ar ri er an d m ak i n g t h e cat al yt i c s t at e m o re acc es s i b l e t o op en M BP. It i s t h er efo r e l i k el y t h at t h e art i fi ci al m em b r an es o f p ro t eo l i p o s o m es fo rm an i n t erm ed i at e en v i ro n m e n t b et ween t h e l o o s e A T P as e re gu l at i o n ob s erv ed i n d et e r gen t an d t i gh t er r e gu l at i o n i n t h e n at i v e cel l m em b ran e. On ce m al t o s e h as en t er e d t h e TM t ran s l o cat i o n p at h wa y, t h e v a can t m al t o s e- b i n d i n g s i t e o f M BP i s re co gn i z ed as a s i gn al t o i ni t i at e ATP h yd ro l ys i s an d m o t i o n s o f t h e M al G s co op l o op an d M al G C -t erm i n u s b ri n g t h e t ran s p o rt e r i n t o a co n f o rm at i o n i n wh i ch ATP can b e h yd ro l yz ed. Fr o m t h e p ers p e ct i v e o f co n fo rm at i o n al d yn am i cs, t h i s 174

186 s t at e l i k el y r efl e ct s an en d p o i n t o f ex t rem e en er get i c s t ab i l i t y. Al t h o u gh e n t rop i c m o v em en t s are l i k el y t o b e m i n i m al, en t h al p i c co n s id erat i o n s h av e n o w s t ab i l i z ed t h e t ran s p o rt er i n a v er y t i gh t co m p l ex. Th i s i s refl ect ed i n t h e h i gh affi n i t y o f op en co n fo rm at i o n M BP fo r t h i s t ran s p o rt er s t at e, as t h e co m p l ex wi l l n o t d is s o ci at e i f ATP h yd ro l ys i s i s i n h i b i t ed [ 16 ]. In p ra ct i ce h o w ev er, t h e co m p l ex h as s i gn i fi ca n t p o t en t i al en er g y i n t h e p h o s p h o an h yd ri d e b o n d s o f e ach b o u n d ATP m o l ecu l e. In t h i s co n fo rm at i o n, h yd ro l ys i s o f o n e o r b o t h ATP m o l ecu l es i s b o t h s p o n t an eo u s an d rap i d. ATP h yd ro l ys i s wi l l d i s rup t t h e M al K d i m er, p ro m o t i n g i t s s ep arat i o n, d es t ab i l i z e t h e cat al yt i c co n fo rm at i o n s u ch t h at i t can rev ert t o th e res t i n g s t at e. Th e s eq u en ce o f co n fo rm at i o n al ch an ges wh i ch ret u rn t h e t ran s p o rt e r co m p l ex t o i t s c yt op l as m i c fa ci n g res t i n g s t at e a re n o t as w ell u n d ers t o o d, p erh ap s o wi n g t o t h e i n s t ab i l i t y o f ea ch d i s cre et s t ag e as t h e co m p l ex pro g res s es t o co n fo rm at i o n s o f i n creas i n g en t rop y. P a ra m agn et i c res o n an ce m eas u rem en t o f t h e s ep ar at i o n o f s p i n - l ab el ed M al K s ub u n i t s h as s u g g es t ed t h at t h e d i s s o ci at i o n o f i n o r gan i c p h o s p h at e cau s es t h e M al K d i m er t o p art i al l y op en [ 1 0 ]. Gi v en t h e r i gi d b o d y d o m ai n ro t at i o n s s een i n t h e TM d o m ai n s b et ween ex i s t i n g M al FG K 2 cr ys t al s t ru ct u res, i t i s l i k el y t h at M al K d i m er s ep arat i o n c au s es t h e co m p l ex t o ad op t a co n fo rm at io n m o re l i k e t h e p re- cat al yt i c t h an t h e cat al yt i c s t at e, b ro ad l y m i rro ri n g ch an ges s een p ri o r t o h yd ro l ys i s [ 7,8,6 ]. Th i s wi l l cau s e t h e i n t e rfa ce wi t h M BP, wh i ch s up p o rt s h i g h -affi n i t y b i n d i n g t o op e n M BP i n t h e cat al yt i c s t at e, t o rev ert t o a p o s i t i o n t h at i s i ns t ead co m p l em en t ar y t o cl o s ed M BP. Bo u n d M BP wi l l b e u n d e r s t rai n fro m t h e t r an s p o r t er an d d ri v en t o w ard s t h e u n l i gan d ed cl o s ed co n fo rm at i o n. Be cau s e t h e cl o s ed fo rm o f M BP i s n o l o n ger s t ab i l i zed b y 175

187 m al t o s e, t h i s rep res en t s a d i s fav o red co n fo rm at i o n, an d wi l l res u l t i n d i s s o ci at i o n o f M BP fro m t h e co m p l ex wh en t h e s t rai n ex ceed s t h e en e rg y o f b i n d i n g. Ob s erv at i o n s i n ch ap t e r 4 wi t h M BP -O an d M BP -O9 5 / 1 71 are co n s i s t en t wi t h t h i s m o d el o f d i s s o ci at i o n. If t h e BM OE cro s s l i n k of t h es e p ro t ei n s c au s es t h em t o res i s t d o m ai n cl o s u re b y M al F GK 2, t h en t h e m o d i fi ed m u t an t s wo u l d res i s t co n v ers i o n o f t h e t ran s p o rt er t o t h e r es t i n g s t at e t o a g re at er d e g ree t h an wt M BP. Th i s co u l d t rap t h e co m p l ex i n a co n fo rm at i o n n ear t o t h e c at al yt i c s t at e s u ffi ci en t l y l o n g fo r ADP t o d i s s o ci at e an d b e r ep l ac e d b y ATP. Th e s ub s t i t u t i on o f ADP fo r ATP wo u l d s h o rt -ci rcu i t t h e cat al yt i c c ycl e an d s t ab i l i z e t h e t ran s p o rt er fo r rep eat ed ATP h yd ro l ys i s. Th es e fi n d i n gs a re v e r y p rel i m i n ar y, req u i ri n g a gre at d e al o f fu rt h e r i n v es t i g at i o n, b u t d o h i n t t h at t h e rev e rs i o n o f t h e t ran s p o rt er t o t h e res t i n g s t at e i n v o l v es co n fo rm at i o n al ch an ges i n M BP, p ro v i d i n g a t i n y wi n d o w i n t o t h at as yet l i t t l e-u n d ers t o o d s t ag e o f t ran s p o rt wh ere m al t o s e p as s es t h e m em b ran e an d i s r el eas e d in t o t h e c yt op l as m. Ob s erv at i o n s o f t h e act i v i t i es o f t h e BM O E cro s s -l i n k ed m u t an t s h ave fo rm ed an i n t ri gu i n g co d a t o t h i s t h es i s al t h o u gh m o re s t u d y i s n ee d ed t o b et t er ch a ract e ri z e t h e eff ect o f m o d i fi cat i o n o n M BP co n fo rm at i o n al d yn am i cs. Th e fi n d i n gs o f t h i s t h es i s d efi n i t i v el y ex p l ai n t he ro l e o f m al t o s e i n re gu l at i n g t ran s p o rt er act i v i t y. W i t h res p ect t o ATP as e act i v at i o n, t h e s ub s t rat e i s n o t fu n ct i o n al l y reco gn i z ed b y t h e TM r e gi o n s o f t h e t r an s p o rt er. In t h e t h i rd ch ap t e r i t wa s ob s erv ed t h at t h e rat e o f ATP h yd ro l ys i s b y s M BP w as n o t al t ere d b y t h e s ub s t i t u t i o n o f m al t o s e fo r s u cro s e. Th i s was ob s er v ed i n s p i t e o f t h e i n ab i l i t y o f t h e TM s ub s t r at e b i n d i n g s i t e t o acco m m o d at e s u cro s e. F u rt h er, i n t h e fo u rt h ch ap t er we ob s e rv ed v er y h i g h rat es o f ATP as e act i v at i o n f ro m M BP -O an d M BP -O9 5 / i n t h e ab s en ce o f m al t o s e. Th es e 176

188 d at a al l i n d i cat e t h at t h e p res en c e o f m al t o s e i s no t an ab s o l u t e req u i r em en t fo r t ran s p o rt er act i v at i o n an d can b e co m p l em en t ed b y mu t at i o n s i n M BP. Th e reco gn i t i o n o f m al t o s e can b e co n s i d ered up s t r eam o f M BP co n fo rm at i o n al ch an ges. Th e ro l e o f m al t o s e i n act i v at i n g M al FGK 2 ATP h yd ro l ys i s i s t h erefo r e ex cl u s i v el y t o s t ab i l i z e co n fo rm at i o n al ch an ges i n M BP. It i s i m p o rt an t t o n o t e t h at t h i s d o es n o t m ean t h at i n t eract i o n s b et we en m al t o s e an d t h e t ran s p o rt er a re i rrel ev an t t o t ran s p o rt e r fu n ct i o n, b u t rat h er t h at t h e y d o n o t co n t ri b u t e t o t h e d eci s i o n t o h yd ro l yz e ATP. In d eed, t h e p res en ce o f a s p eci fi c m al t o s e-b i n d i n g s i t e wi t h i n t h e TM r egi o n s o f M al FG K 2 t es t i fi es t o t h e i m p o rt an ce o f co n t act s b et ween s ub s t r at e an d t r an s po rt er [ 6,1 5 ]. It i s l i k el y t h at co n t act s at t h i s s i t e p l a y a ro l e i n en s u ri n g t h at ATP h yd rol ys i s res u l t s i n s ub s t rat e t r an s p o rt rat h e r t h an i n i n i t i at i n g ATP h yd ro l ys i s i n res p o n s e t o t h e av ai l ab i l i t y o f s ub s t rat e. C o n s i s t en t wi t h t h i s t h eo r y, a r ec en t s t u d y h as fo u n d t h at m u tat i o n s wh i ch p rev en t t h e p as s a ge o f s ub s t rat e i n t o t h e t ran s l o cat i o n p at h wa y p r ev en t t r an s p o rt wi t h o u t p rev en t i n g ATP h yd ro l ys i s [ 1 7 ]. It t h ere f o re ap p e ars t h at ATP b i n d i n g an d h yd ro l ys i s i s i n f l u en ced b y i n t eract i o n s b et we en m al t o s e an d M BP an d b et we en M BP an d t h e TM re gi o n s, wh i l e t h e en er g y r el eas ed fro m t h at h yd ro l ys i s i s d i rect ed t o ward s s ub s t rat e t r an s p o rt b y i n t eract i o n s b et we en m al t o s e an d t h e TM re gi o n s o f t h e t ran s p o rt e r. M o re s t u d i es ar e n eed ed t o fu l l y d ev el op an d t es t th i s m o d el o f act i v i t y. Du ri n g t h e wo rk o f t h i s t h es i s, w o rk was b e gu n o n a co u rs e o f ex p eri m en t s i n t en d e d t o ob s erv e t h e eff ect s o f al t e ri n g t h e rel at i v e s t ab i l i t i es o f co m p et i n g M al F GK 2 co n fo rm at i o n s. Ti m e co n s t rai n t s h av e u n fo rt u n at el y p rev en t ed t h i s wo rk fro m b ei n g co m p l et ed. P ri o r t o t h e s u s p en s i o n o f t h i s wo rk, n i n e m u t an t fo rm s o f t h e t ran s p o rt er w ere cl o n ed wi t h m u t at i o n s i n M al F an d M al G k n o w n t o cau s e a M BP i n d ep e n d en t ph en o t yp e (ap p en d i x 3 )[ 1 8 ]. 177

189 M BP i n d ep en d en t M al F GK 2 m u t an t s are ab l e t o t ran s p o rt m al t o s e wi t h o u t b ei ng s t i m u l at ed b y M BP, as i n d i cat ed b y t h ei r ab i l i t y t o co m p l em en t g ro wt h o n m al t o s e i n M BP d efi ci en t E. coli [ 19 ]. In t h i s res p e ct t h e y re s em b l e ABC ex p o rt com p l ex es, wh i ch act d i rect l y o n s ub s t rat e. In t ri gu i n gl y, t h es e m u ta n t s ex h i b it a l ev el o f co n s t i t u t i v e ATP h yd ro l ys i s t h at i s i n p rop o rt i o n t o t h ei r rat e o f M B P i n d ep en d en t m al t o s e t r an s p o rt [ 1 8 ]. Th i s i s rem i n i s cen t o f t h e p h en o t yp e b ro u gh t o n b y d et er gen t s o l ub i l i s at i o n. Th e n i n e m u t an t s s el ect ed an d cl o n ed d i s p l a y a v ar yi n g d e gr ee o f co n s t i t u t i v e ATP as e act i v i t y acro s s a wi d e ran g e. In t h e fu t u re, t h es e m u t a n t t ran s p o rt ers c an b e t est ed i n l i p o s o m es an d d et er gen t as t h e wi l d -t yp e t ran s p o rt e r h as b een t es t ed. Th e p r e s en t ed m o d el i n d i cat es t h at, l i k e wt M al FGK 2 i n t h e d et er g en t s o l ub i l i z ed p h as e, i n d ep en d en t t ran s p o rt ers h a v e cat al yt i c s t at es t h at are m o r e re ad i l y a cces s i b l e t o op en M B P. Th at t h es e t ran s p o rt e r s can act d i rect l y o n m al t o s e i s an i n d i cat i o n t h at t h e y c an a d op t t h e p eri p l as m i c fa ci n g ATP as e cat al yt i c co n fo rm at i o n w i t h o u t M BP, wh i ch i n d i cat es t h at m u t at i o n h as l o wered at l eas t t h e fi rs t en er g et i c b ar ri er. Tran s p o rt ers t h at d em o n s t rat e l o o s en ed r e gu l at i o n b y an i n creas e i n u n co up l ed A TP as e act i v i t y s h o u l d h a v e st ab i l i z ed ATP as e cat al yt i c s t at es, as i n d i cat ed b y gre at er ATP as e s t i m u l u s b y op en M B P wi t h o u t m al t o s e. A co rrel at i o n s h o u l d ex i s t b et ween t h e rat e o f co n s t i t u t i v e u n co up l ed ATP h yd ro l ys i s an d t h e d e gre e o f fu rt h er ATP as e s t i m u l at i o n b y t h e op en u n l i g an d e d fo rm o f M BP. S u ch a f i n d i n g wo u l d s up p o rt t h e p rop o s ed m o d el b y i n d i c at i n g t h at d i s - re gu l at i o n o f t h e m u t an t t ran s p o rt ers, an d t h e ac co m p an yi n g i n d ep en d en ce f ro m M BP, i s carri ed o u t b y a s t ab i l i z at i o n o f t ran s p o rt er co n fo rm at i o n s t h at are n o rm al l y d i s fav ou red i n t h e ab s en ce o f b i n d i n g p ro t ei n. Fu rt h e r t es t i n g u s i n g co n fo rm at i o n al m u t an t s s u ch as M BP -C co u l d fu rt h er 178

190 d i s t i n gu i s h t h e i m p act o f M BP i n d ep en d en t m u t at i o n s o n each s t a g e o f t h e cat al yt i c c yc l e. Th e fi n d i n gs o f t h i s wo rk em p h as i z e t h e co m p l ex i ty o f t h e i n t e ra ct i o n b et we en b i n d i n g p ro t ei n an d co gn at e ABC t r an s p o rt er. C o up l ed ATP h yd ro l ys i s an d s ub s t rat e t ran s p o rt ar e n o t s i m p l y t ri g ge red b y t h e as s o ci at i o n o f a co m p l em en t a r y b i n d i n g s u rf ace, b u t ari s e fro m a m u l t i s t a ge s h ep h erd i n g o f t h e t ra ns p o rt er t h ro u gh t h e c at a l yt i c c ycl e. M al FGK 2 ex i s t s i n an en s em b l e o f co m p et i n g co n fo rm at i o n s wh i ch re gu l at e ATP h yd ro l ys i s b y t h ei r r el at i v e s t ab i l i t y. Bo t h co n for m at i o n s o f t h e m al t o s e b i n d i n g p ro t ei n s erv e t o p ro m o t e ATP h yd ro l ys i s b y m o d u l at i n g t h e rel at i v e s t ab i l i t y o f t h e s e co n fo rm at i o n s. Us i n g a s eri es o f m u t an t b i n d i n g p ro t ei n s i t h as b een d em o n s t rat ed t h at b o t h op en an d cl o s ed b i n d i n g p ro t ei n co n fo rm at i o n s p ro m o t e ATP h yd ro l ys i s t h ro u gh t h ei r i n t eract i o n s wi t h t h e t ran s p o rt e r. It h as als o b een d et e rm i n ed t h at t h e t ran s p o rt ed s ub s t rat e d o es n o t i n i t i at e ATP h yd ro l ys i s b y i n t er act i o n s wi t h t h e t ran s p o rt er, b u t i n s t ead act s t o s t ab i l i z e t h e cl o s ed co n fo rm at i o n o f M BP t o en ab l e i t t o act i v at e t h e t ran s p o rt er. 179

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193 Ap p en d i x 1 C o o m as s i e St ai n ed 1 5 % S DS -P AGE gel o f P r ep ar ed Bi n d i n g P ro t ei n s 182

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