Functional Role of Loop 2 in Myosin V

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1 Biochemistry 2004, 43, Functionl Role of Loop 2 in Myosin V Christopher M. Yengo nd H. Lee Sweeney* Deprtment of Physiology, UniVersity of PennsylVni School of Medicine, Phildelphi, PennsylVni ReceiVed August 22, 2003; ReVised Mnuscript ReceiVed Decemer 3, 2003 ABSTRACT: Myosin V is moleculr motor tht is cple of moving processively long ctin filments. The kinetics of monomeric myosin V contining single IQ domin (MV 1IQ) differ from nonprocessive myosin II in tht ctin ffinity is higher, phosphte relese is extremely rpid, nd ADP relese is rtelimiting. We generted two mutnts of myosin V y ltering loop 2, surfce loop in the ctin-inding region thought to lter ctin ffinity nd phosphte relese in myosin II, to determine the role tht this loop plys in the kinetic tuning of myosin V. The loop 2 mutnts ltered the pprent ffinity for ctin (K ATPse ) without ltering the mximum ATPse rte (V MAX ). Trnsient kinetic nlysis determined tht the rte of inding to ctin, s well s the ffinity for ctin, ws dependent on the net positive chrge of loop 2, while other steps in the ATPse cycle were unchnged. The mximum rte of phosphte relese ws unchnged, ut the ffinity for ctin in the M ADP Pi-stte ws drmticlly ltered y the muttions in loop 2. Thus, loop 2 is importnt for llowing myosin V to ind to ctin with reltively high ffinity in the wek inding sttes ut does not ply direct role in the product relese steps. The ility to mintin high ffinity for ctin in the wek inding sttes my prevent diffusion wy from the ctin filment nd increse the degree of processive motion of myosin V. Myosins mke up lrge superfmily of motor proteins tht re cple of using the chemicl energy from ATP hydrolysis to power the directed movement on ctin filments nd function in wide vriety of cellulr processes from muscle contrction to orgnelle trnsport (1). During the ctomyosin ATPse cycle myosin shifts etween ctindetched (wek-inding) nd ctin-ttched sttes (stronginding), nd force genertion occurs through conformtionl chnge in myosin during the trnsition from the wekto the strong-ctin inding sttes (reviewed in ref 2). Interestingly, the overll structure of myosin proteins ppers to e quite conserved, while their iochemicl nd kinetic properties re quite divergent. It hs een proposed tht sequence vriility in the two surfce loops of myosin, which re susceptile to proteolysis nd divide myosin into three domins (25, 50, nd 20 kd, Figure 1), my ply role in kineticlly tuning prticulr myosin to perform specific cellulr functions (3-6). In the current study, we exmine the role of loop 2, surfce loop in the ctin-inding region of myosin, in kineticlly tuning myosin V, nonmuscle myosin tht functions s n orgnelle trnsporter. Myosin V hs severl unique iochemicl fetures tht llow it to move processively long ctin (7), tke multiple steps long ctin without diffusing wy, nd function s n orgnelle trnsporter (8). The kinetics of myosin V re This work ws supported y Ntionl Institute of Helth Grnt AR35661 (to H.L.S.). * To whom correspondence should e ddressed: Deprtment of Physiology, A400 Richrds Bldg., University of Pennsylvni School of Medicine, Phildelphi, PA Tel: Fx: E-mil: lsweeney@mil.med.upenn.edu. A Ntionl Institute of Helth NRSA Postdoctorl Fellow. Current ddress: Deprtment of Biology, University of North Crolin t Chrlotte. FIGURE 1: Alignment of myosin loop 2 sequences. The lignment of loop 2 sequences from chicken myosin V (MV), smooth muscle myosin II (Sm), skeletl muscle myosin II (Sk), nd Dictyolstelium myosin II (Dd) is shown, long with the overll chrge in prentheses nd size of the loop 2 from ech isoform. The lysine residues in myosin V tht were sustituted with lnines nd the four residues tht were deleted re underlined. Also the smooth muscle myosin lysines tht were sustituted with lnines re indicted in itlics. The lignment ws dpted from ref 1. different from nonprocessive muscle myosin II in tht ADP relese in myosin V is slow nd rte-limiting, while phosphte relese is fst (9-11). This llows myosin V to populte the strong-inding sttes for greter frction of its ATPse cycle nd increse its duty rtio, frction of ATPse cycle tht myosin is strongly ound to ctin. In previous report, we determined tht myosin V hs higher ffinity for ctin in the wek inding sttes (12) thn nonprocessive clss II myosins, which my lso e n importnt spect of its ility to move processively. Thus, the current study is focused on exmining the structurl spects of myosin V tht give rise to its high ffinity for ctin. One unique structurl feture of myosin V is tht it hs lrge nd highly chrged surfce loop, commonly referred /i035510v CCC: $ Americn Chemicl Society Pulished on We 02/12/2004

2 2606 Biochemistry, Vol. 43, No. 9, 2004 Yengo nd Sweeney to s loop 2, in its ctin-inding region (see ref 1 for comprison of myosin isoforms). Studies exmining the role of loop 2 in myosin II hve determined tht vriility in the length nd numer of chrged residues in this loop cn lter myosin s ffinity for ctin, s well s its enzymtic ctivity (4, 13-17). In ddition, study on smooth muscle myosin demonstrted tht sustituting two lysine residues in loop 2 completely locked the ctin-ctivted ATPse ctivity of this myosin, ut 16 mino cid deletion of the loop tht retined the criticl lysine residues ctully incresed smooth muscle myosin s ffinity for ctin (18). Alignment of loop 2 of myosin V with other myosin isoforms demonstrtes unique six mino cid insert in the N-terminl region s well s severl positively chrged residues in the C-terminl prt of the loop (Figure 1). Therefore, it is uncler whether the unique fetures of loop 2 of myosin V re importnt for incresing ffinity for ctin or controlling ctin ctivtion of product relese or oth. We exmined the role of loop 2 in myosin V y performing complete kinetic chrcteriztion of two mutnts of myosin V with modified loop 2 sequences. We generted one mutnt of myosin V with single IQ inding motif (MV 1IQ) tht hd three lysine residues (residues 630, 633, 634) sustituted with lnines (LP2AAA MV 1IQ) in the sme region s the lysines tht were sustituted with lnines in the smooth muscle myosin study (18). We lso generted mutnt with four mino cids deleted from the unique insert in the N-terminl region of loop 2 (residues Asp595, Glu596, Glu597, Lys598). In ddition, we further exmined the kinetic properties of monomeric version (sufrgment 1, S1) of smooth muscle myosin mutnt with two lysine residues sustituted with lnines, which ws previously exmined in dimeric hevy meromyosin (HMM) ckground (18), to directly compre it to our myosin V loop 2 mutnts. Our results suggest structurl role for loop 2 in llowing myosin V to ind to ctin with reltively high ffinity in the wek inding sttes compred to myosin II, while not ltering other steps in the ctlytic cycle of myosin V. EXPERIMENTAL PROCEDURES Regents. All regents were the highest purity commercilly ville. ATP ws prepred fresh from powder (Roche Moleculr Biochemicls, 99.7% pure y HPLC (dt not shown). N-Methylnthrniloyl (mnt)-leled ADP nd ATP were prepred s descried (19). ATP nd ADP concentrtions were determined y sornce t 259 nm using ɛ 259 of M -1 cm -1. Nucleotides were prepred prior to use in the presence of equimolr MgCl 2. Myosin cdna Construction nd Protein Expression nd Purifiction. Site-directed mutgenesis ws performed on construct of chicken myosin V contining single IQ motif (WT MV 1IQ) (residues 1-792). We sustituted three lysine residues in loop 2 (630, 633, nd 634) with lnines to generte the LP2AAA MV 1IQ construct, nd we deleted four residues from the N-terminl portion of loop 2 (residues Asp595, Glu596, Glu597, nd Lys598) to generte the DEEK MV 1IQ construct. The culovirus system ws used to express the myosin V nd smooth muscle myosin constructs, which contined C-terminl FLAG tg for purifiction purposes (5). All myosin V constructs were coexpressed with the essentil light chin LC-1s (9), nd the smooth muscle myosin sufrgment 1 (SmS1) constructs were coexpressed with the essentil nd regultory light chins (18). The purity ws greter thn 95% sed on Coomssie stined SDS gels. Myosin concentrtions were determined using the Bio-Rd microplte ssy (5). Actin ws purified from rit skeletl muscle using n cetone powder method (20) nd gel filtered. Pyrene ctin ws generted y leling ctin with pyrene iodocetmide (Moleculr Proes) s descried (21). All experiments with myosin V were performed in KMg50 uffer (50 mm KCl, 1 mm EGTA, 1 mm MgCl 2,1mM DTT, nd 10 mm imidozole-hcl, ph 7.0, 25 C) nd those with smooth muscle myosin in 20/20 uffer (20 mm MOPS, ph 7.0, 20 mm KCl, 1 mm EGTA, 5 mm MgCl 2, nd 1 mm DTT) t 25 C. Since SmS1 hs weker ffinity for ctin, it ws necessry to perform the ctin-inding nd stedy-stte ATPse experiments in 20/20 uffer. Stedy-Stte ATPse ActiVity of MV 1IQ. Stedy-stte ATP hydrolysis y MV 1IQ LC-1s ( nm) or SmS1 (200 nm) in the presence nd sence of ctin (0-40 µm) ws exmined using the NADH-linked ssy (9) with finl MgATP concentrtion of 1 mm. Stopped-Flow Mesurements nd Kinetic Modeling. Trnsient kinetic experiments were performed in Applied Photophysics (Surrey, U.K.) stopped-flow with ded-time of 1.2 ms. Tryptophn fluorescence ws mesured y exciting the smple t 295 nm, nd the emission ws mesured using 320 nm long pss filter. Pyrene ctin ws excited t 365 nm nd mnt-leled nucleotides were excited t 295 nm, nd their emission ws mesured using 400 nm long pss filter. The kinetics of phosphte relese were mesured using phosphte-inding protein covlently leled with N-[2-(1- mleimidyl)ethyl}-7-(diethylmino)coumrin-3-croxmide (MDCC-PBP) (generously provided y Steven Rosenfeld, University of Alm) (22). The fluorescence of MDCC-PBP, which increses severlfold in the presence of inorgnic phosphte (22, 23), ws excited t 425 nm, nd the emission ws mesured through 435 nm long pss filter. Phosphte relese ws mesured with sequentil mix experiment in which 2 µm MV 1IQ ws mixed 10 µm ATP nd ged for 1stollow MV 1IQ to ind nd hydrolyze the ATP, nd then the MV ADP Pi complex ws mixed with ctin (0-45 µm), 5 µm MDCC-PBP, nd 2 mm ADP (24) (finl concentrtions fter mixing 0.5 µm MV 1IQ, 2.5 µm ATP, 5 µm MDCC-PBP, nd 2 mm ADP). All solutions were preincuted with 7-methylgunosine (0.2 mm) nd purine nucleoside phosphorylse (0.2 units ml -1 ) to remove ckground phosphte. Nonliner lest-squres fitting of the dt ws done with softwre provided with the instrument or Kliedgrph (Synergy Softwre, Reding, PA). Uncertinties reported re stndrd error of the fits unless stted otherwise. Kinetic modeling nd simultions were performed using the rection scheme shown in Scheme 1), which hs een used in recent kinetics studies of myosin V (9-12). In this scheme, myosin, ctin, nd ctomyosin re represented y M, A, nd AM, respectively. The rte nd equilirium constnts re leled on the sis of the rection proceeding from left to right nd those etween the ctin-ssocited nd

3 Kinetics of Myosin V Loop 2 Mutnts Biochemistry, Vol. 43, No. 9, Scheme 1 Tle 2: Rte nd Equilirium Constnts for ATP Binding nd Hydrolysis construct K 1k +2 k +3 + k -3 K 1 k +2 (µm -1 s -1 ) (s -1 ) (µm -1 s -1 ) K 1 (µm) k +2 (s -1 ) WT MV 1IQ 1.6 ( ( ( ( ( 70 LP2AAA MV 1IQ 2.6 ( ( ( ( ( 20 DEEK MV 1IQ 1.9 ( ( ( ( ( 89 Mesured with intrinsic tryptophn fluorescence in the sence of ctin. Mesured with pyrene ctin fluorescence. FIGURE 2: Stedy-stte ATPse of MV 1IQ constructs. The ATPse rte of WT MV 1IQ (), LP2AAA MV 1IQ ([), nd DEEK MV 1IQ (0) were mesured with the NADH-coupled ssy nd plotted s function of ctin concentrtion (myosin concentrtion 100 nm). The dt were fit to the Michelis-Menton eqution to determine the V MAX (mximum rte of ATP turnover) nd K ATPse (ctin concentrtion t which there is hlf-mximl ctivtion). The men ATPse rte t ech ctin concentrtion determined from lest two seprte myosin V preprtions ws plotted nd fit to the Michelis-Menton eqution to determine V MAX nd K ATPse. The error rs represent stndrd devitions of the men ATPse rte t ech ctin concentrtion. The dt re summrized in Tle 1. Tle 1: Summry of Stedy-Stte ATPse Results construct V MAX (mol of Pi s -1 (mol of myosin) -1 ) K ATPse (µm) WT MV 1IQ 14.1 ( ( 0.6 LP2AAA MV 1IQ 13.6 ( ( 1.4 DEEK MV 1IQ 12.7 ( ( 0.1 Mximum rte of ATP turnover s determined from fitting the dt in Figure 2 to the Michelis-Menton eqution. Actin concentrtion t which the ATPse rte is one-hlf of the mximl rte lso determined from fitting the dt in Figure 2 to the Michelis-Menton eqution. -dissocited steps proceeding in the dissocited direction. The min flux of the rection pthwy is shown in old. RESULTS Stedy-Stte ATPse Assys. The stedy-stte ATPse rtes of WT nd mutnt MV 1IQ were plotted s function of ctin concentrtion nd fit to the Michels-Menton eqution to determine the mximum rte of ATP turnover (V MAX ) nd ctin concentrtion t which the ATPse rtes were one-hlf the mximl rte (K ATPse ) (Figure 2, Tle 1). The verge ATPse rte t ech ctin concentrtion determined from lest two seprte myosin V preprtions ws plotted nd fit to the Michelis-Menton eqution to determine V MAX nd K ATPse. The K ATPse of LP2AAA MV 1IQ ws incresed 4-fold (8.8 ( 1.4 µm) nd the K ATPse of DEEK MV 1IQ ws reduced 3.7-fold (0.6 ( 0.1 µm) compred WT MV 1IQ (2.2 ( 0.6 µm), while the V MAX s of oth mutnts were very similr to WT MV 1IQ (13.6 ( 1.0, 12.7 ( 0.3, 14.1 ( 1.1 s -1, respectively). The stedy-stte ATPse of monomeric LP2AA SmS1 t 60 µm ctin (0.09 s -1 ) ws similr to the sl ATPse rte in the sence of ctin (0.08 s -1 ), while the WT SmS1 ws ctivted severlfold (0.11 nd 0.33 s -1, respectively) t 37 C in 20/20 uffer. ATP Binding nd Hydrolysis. The tryptopn fluorescence enhncement of MV 1IQ cn e used to monitor the rtes of oth ATP inding nd hydrolysis (9, 12). Plots of the rte of the tryptophn fluorescence increse s function of ATP concentrtion were fit to hyperol, nd the extrcted rte nd equilirium constnts for ATP inding nd hydrolysis re shown in Tle 2. The second-order rte constnt for ATP inding to MV 1IQ, determined from the initil slope of the hyperol, ws very similr in the WT nd mutnt MV 1IQ constructs (K 1 k µm -1 s -1 ). In ddition, the effective rte constnt of hydrolysis, determined from the mximum rte of the tryptophn fluorescence signl, ws lso very similr in the WT nd mutnt MV 1IQ constructs (k +3 + k s -1 ). ATP-Induced Dissocition of cto-mv 1IQ. The ATPinduced dissocition of MV 1IQ from ctin ws monitored with pyrene ctin fluorescence s descried (9). Pyrene ctin is quenched when myosin is strongly ound to ctin nd recovers when ATP-inding induces formtion of the wekly ound ctomyosin sttes. The dt were fit to two exponentils with the fst phse modeled s the rte of ATP-induced dissocition nd the slow phse s the rte of ADP relese. The fst phse consisted of 90% of the totl mplitude, while the slow phse ws pproximtely 10% of the totl mplitude. Thus, lthough the ctomyosin smple ws treted with pyrse, 10% of the MV 1IQ still hd ADP ound prior to mixing with ATP. The rte of the slow phse ws similr to the rte of mntadp relese nd the V MAX from the stedystte ATPse ssy. The rte of the fst phse of the pyrene fluorescence recovery ws plotted s function of ATP concentrtion nd fit to hyperol (Figure 3) to determine the rte nd equilirium constnts for ATP inding to cto- MV 1IQ (Tle 2). The rtes nd equilirium constnts for ATP-induced dissocition of ctomyosin V 1IQ were very similr in the WT nd LP2AAA MV 1IQ constructs

4 2608 Biochemistry, Vol. 43, No. 9, 2004 Yengo nd Sweeney FIGURE 3: ATP-induced dissocition of cto-mv 1IQ. In pnel A, the ATP-induced dissocition of cto-mv 1IQ (0.15 µm) (k os ) nd the ATP concentrtion dependence ws determined for WT MV 1IQ (), LP2AAA MV 1IQ ([), nd DEEK MV 1IQ (0). Points re the verge of 1-3 trnsients, nd error rs represent the stndrd errors of the fit to the verged trnsient. Pnel B presents time courses of ATP-induced dissocition of MV 1IQ constructs from pyrene ctin. The fst phses of the time courses were 642 ( 29, 789 ( 36, nd 585 ( 39 for () LP2AAA MV 1IQ, () DEEK MV 1IQ, nd (c) WT MV 1IQ, respectively, nd (d) the uffer control. Finl rection conditions were s follows: 0.15 µm pyrene ctomyosin nd 2 mm MgATP. The rte nd equilirium constnts determined from the hyperolic fits of the dt nd stndrd error from the fits re shown in Tle 2. FIGURE 4: Binding of MV 1IQ to pyrene ctin filments. The rte (k os )ofwtmv1iq(), LP2AAA MV 1IQ ([), or DEEK MV 1IQ (0) inding to pyrene ctin filments ws determined y monitoring the rte of pyrene fluorescence quenching upon mixing MV 1IQ with 10-fold excess pyrene ctin. Pnel A shows tht the rtes of pyrene ctin inding in the presence of 0.4 mm ADP were linerly dependent on ctin concentrtion. Pnel B shows tht the rtes of pyrene ctin inding in the sence of nucleotide (pyrsetreted) were lso linerly dependent on ctin concentrtion. Points re the verge of 1-3 trnsients, nd error rs represent the stndrd errors of the fits. The rte nd equilirium constnts determined from the liner fits to the dt re shown in Tle 3. Finl rection conditions were s follows: ctin concentrtion s indicted, MV 1IQ concentrtion 10-fold lower thn the ctin, nd 0.4 mm ADP. (K 1 ) 477 ( 44 nd 718 ( 51 µm, respectively, k +2 ) 771 ( 70 nd 853 ( 20 s -1, respectively, nd K 1 k µm -1 s -1 ; see Tle 2). However, the DEEK MV1IQ construct hd similr second-order rte constnt nd equilirium constnt for ATP-inding (K 1 k +2 ) 1.9 ( 0.4 µm -1 s -1 nd K 1 ) 548 ( 125 µm, respectively) s descried ove ut displyed slightly fster mximum rte of ATP-induced dissocition (k +2 ) 1080 ( 89 s -1 ) from pyrene ctin thn WT MV 1IQ. Binding to Pyrene Actin Filments. MV 1IQ inding to ctin ws mesured y monitoring the rte of pyrene ctin fluorescence quenching upon mixing MV 1IQ with 10-fold excess pyrene ctin in the presence nd sence of ADP. The rte of pyrene ctin fluorescence quenching fit well to single exponentil nd ws linerly dependent on the ctin concentrtion, in the rnge of ctin concentrtions mesured (1-10 µm ctin), for WT, LP2AAA, nd DEEK MV 1IQ (Figure 4). A 5-fold reduction in the second-order rte constnt for inding to pyrene ctin in the presence of ADP ws oserved for the LP2AAA MV 1IQ (k -10 ) 0.9 ( 0.1 µm -1 s -1 ) construct compred to WT-MV 1IQ (k -10 ) 5.5 ( 0.1 µm -1 s -1 ), while 3-fold increse in the secondorder rte constnt ws oserved for DEEK MV 1IQ (k -10 ) 14.6 ( 0.4 µm -1 s -1 ). Dissocition from pyrene ctin, mesured y mixing MV 1IQ ound to pyrene ctin with 20-fold excess unleled ctin, ws 0.08 s -1 for WT nd LP2AAA MV 1IQ nd 0.04 s -1 for DEEK MV 1IQ. We lso mesured the rte of MV 1IQ inding to pyrene ctin s descried ove ut in the sence of nucleotide. The second-order rte constnt for LP2AAA MV 1IQ (k -6 ) 38 ( 1 µm -1 s -1 ) inding to pyrene ctin ws reduced 3-fold compred to WT MV 1IQ (k -6 ) 90 ( 1 µm -1 s -1 ), while the second-order rte constnt for DEEK MV 1IQ (k -6 ) 100 (1 µm -1 s -1 ) inding to ctin ws similr to WT MV 1IQ. We lso nlyzed the ctin inding properties of smooth muscle myosin II loop 2 mutnt (LP2AA SmS1) (18) tht is similr to our LP2AAA myosin V mutnt. We mesured pyrene ctin inding in the presence of ADP s descried ove for WT nd LP2AA SmS1 (Figure 5). We mesured the rte of SmS1 inding to ctin, which ws dependent on the ctin concentrtion in hyperolic mnner s descried previously (25) nd llowed us to otin the mximum rte of inding to ctin in the presence of ADP. Although it is

5 Kinetics of Myosin V Loop 2 Mutnts Biochemistry, Vol. 43, No. 9, FIGURE 5: Binding of SmS1 to pyrene ctin filments. The rte (k os ) of WT SmS1 () nd LP2AA SmS1 ([) inding to ctin in the presence of ADP ws determined s in Figure 4 except experiments were performed in 20/20 uffer. Points re the verge of 1-3 trnsients, nd error rs represent the stndrd errors of the fits. The rte nd equilirium constnts determined from the hyperolic fits re listed in the Results. possile tht the ctin inding rtes of MV 1IQ my lso sturte in lower ionic strength 20/20 uffer, we were unle to perform the experiment ecuse of protein ggregtion prolems of MV 1IQ in 20/20 uffer. The LP2AA SmS1 construct hd 2-3-fold reduced mximum rte of inding to ctin compred to WT SmS1 (8.3 ( 2.3 nd 20.3 ( 1.7 s -1, respectively), nd the ctin concentrtion t which hlfmximl sturtion occurs ws reduced 3-fold compred to WT SmS1 (6.1 ( 1.7 nd 19.4 ( 11.6 µm, respectively). The second-order inding constnt for LP2AA SmS1 inding to ctin in the presence of ADP ws reduced nerly 10-fold compred to WT SmS1 (0.4 ( 0.1 nd 3.3 ( 0.7 µm -1 s -1, respectively). ADP Relese from cto-mv 1IQ. The rte of ADP relese from MV 1IQ in the presence of ctin ws determined y monitoring mnt-adp dissocition from cto-mv 1IQ upon mixing with excess unleled ADP (Figure 6). The rtes of ADP relese from cto-wt nd -LP2AAA were very similr (15.1 ( 0.2 nd 15.5 ( 0.1, respectively), while ADP relese from cto-deek MV 1IQ ws slightly incresed (19.6 ( 0.8 s -1 ) (Tle 3). We lso mesured the rte of ADP relese from cto-deek MV 1IQ y competition with ATP-induced dissocition from pyrene ctin s descried ove (the slow phse of the iexponentil fit to the dt), which gve rte (12 ( 0.8 s -1 ) similr to the V MAX from the stedy-stte ATPse ssy. The ADP relese rtes for WT nd LP2AAA MV 1IQ, determined s descried ove, were quite similr to the mnt-adp relese rtes (14 ( 1 nd 17 ( 2, respectively). Actin-ActiVted Phosphte Relese. The relese of phosphte from MV 1IQ ws mesured y performing sequentil mix experiment s descried in Experimentl Procedures. The fluorescence trces consisted of fst single exponentil phse (modeled to e the phosphte relese rte) followed y liner phse (modeled to e the stedy-stte turnover rte). The rte of phosphte relese ws hyperoliclly dependent on ctin concentrtion nd reched mximum tht ws similr in the WT, LP2AAA, nd DEEK MV 1IQ constructs (k 4 ) 110 ( 10, 153 ( 75, nd 101 ( 9s -1, respectively) (Figure 7A). However, the ctin concentrtion t which hlf-mximl sturtion ws chieved (K 9 ) ws incresed 5-fold in the LP2AAA (51 ( 38 µm) FIGURE 6: ADP relese from cto-mv 1IQ. The rte of ADP dissocition from cto-mv 1IQ ws mesured y mixing cto-mv 1IQ (1 µm) ound to mnt-adp (10 µm) with excess unleled ADP (2 mm) (ll concentrtions listed re finl). The rte of the fluorescence decrese ws fit to single exponentil. The time courses for the mnt-adp relese from WT (A), LP2AAA (B), nd DEEK MV 1IQ (C) in the presence of ctin nd their corresponding single-exponentil fits (15.1 ( 0.2, 15.5 ( 0.1, nd 19.6 ( 0.8, respectively) re shown. The fluorescence trces re the verge of 1-3 trnsients. compred to WT MV 1IQ (9 ( 2 µm), while DEEK MV 1IQ (3 ( 1 µm) ws reduced nerly 5-fold compred to WT MV 1IQ. A slight vriility in myosin concentrtion my hve ltered the liner phse of the phosphte relese trnsients (Figure 7B). DISCUSSION We hve determined tht loop 2 of myosin V plys n importnt role in myosin V s ility to ind ctin with reltively high ffinity in the wek inding sttes. The modifictions tht we mde to loop 2 of myosin V ffected the ctin-inding kinetics without significntly ltering other steps in the ctomyosin V ATPse cycle. In contrst, similr

6 2610 Biochemistry, Vol. 43, No. 9, 2004 Yengo nd Sweeney Tle 3: Summry of Rte nd Equilirium Constnts for Actin Binding nd Product Relese construct k -10 (µm -1 s -1 ) k +10 K 10 (s) -1 (µm) k -6 (µm -1 s -1 ) WT MV 1IQ 5.5 ( ( ( 1 9( ( ( 0.2 LP2AAA MV 1IQ 0.9 ( ( ( 1 51( ( ( 0.1 DEEK MV 1IQ 14.6 ( ( ( 1 3( ( ( 0.8 K 9 (µm) Mesured with pyrene ctin. Mesured with phosphte-inding protein. c Mesured with mnt-adp. k +4 (s -1 ) k -5 c (s -1 ) FIGURE 7: Actin-ctivted phosphte relese from MV 1IQ constructs. The rte (k os ) of phosphte relese ws mesured y performing sequentil mix experiment nd using phosphteinding protein s n indictor of inorgnic phosphte production s descried in Experimentl Procedures. In pnel A, the rte of phosphte relese ws plotted s function of ctin concentrtion nd fit to hyperol. The rte nd equilirium constnts determined from the hyperolic fits to the dt re shown in Tle 3. Points re the verge of 1-3 trnsients, nd error rs represent the stndrd errors of the fits. Pnel B shows the time course of phosphte relese from LP2AAA (lower trce), DEEK (upper trce), nd WT MV 1IQ (middle trce) constructs in the presence of 45 µm ctin. The time courses were fit to single exponentil with slope (the vlues for the single-exponentil fits were 65 ( 8, 89 ( 2, nd 92 ( 2,for LP2AAA, DEEK nd WT MV 1IQ, respectively). Finl rection conditions were s follows: 0.5 µm MV 1IQ, 5 µm PBP, indicted ctin concentrtion, nd 2 mm ADP. muttion in smooth muscle myosin completely locked its ctin-ctivted ATPse ctivity nd in vitro motility (18). Our results indicte tht the net positive chrge of loop 2 nd not necessrily the size of the loop medites the ffinity of myosin V for ctin in the wek inding sttes. Thus, the current study provides structurl mechnism for how myosin V contins high ffinity for ctin in the wek inding sttes. Kinetics of MV 1IQ Loop 2 Mutnts. The results of the stedy-stte ATPse ssys clerly demonstrte tht the modifictions tht we mde to loop 2 of myosin V ltered the K ATPse without chnging the V MAX. Sustitution of three lysines to lnines in loop 2 (LP2 AAA MV 1IQ) incresed the K ATPse, indicting weker ffinity for ctin, nd the deletion of four residues, three negtively chrged nd one positively chrged, in loop 2 (DEEK MV 1IQ) reduced the K ATPse, indicting higher ffinity for ctin. Thus, the muttions mde to loop 2 ffected the ffinity for ctin without ltering the rte-limiting step (ADP relese). We performed complete kinetic nlysis of the MV 1IQ loop 2 mutnts to determine wht steps in the ATPse cycle were ltered compred to WT MV 1IQ. The rtes of ATP inding nd hydrolysis, mesured with intrinsic tryptophn fluorescence, were quite similr in the mutnt nd WT MV 1IQ constructs (Tle 2). In ddition, the rtes of ATPinduced dissocition, monitored with pyrene ctin fluorescence, were lso very similr in the mutnt nd WT MV 1IQ constructs. A slight increse in the mximum rte of dissocition (k +2 ) ws oserved with DEEK MV 1IQ. Perhps rerrngement of loop 2 is necessry to llow dissocition from ctin, nd the shorter loop cn more efficiently undergo this conformtionl chnge. Our results demonstrte tht modifying the structure of loop 2 drmticlly lters the ctin inding kinetics of myosin V. We directly mesured MV 1IQ inding to pyrene ctin in the presence nd sence of ADP. As demonstrted previously (9), the rte of myosin V inding to pyrene ctin ws linerly dependent on ctin concentrtion in oth the presence nd sence of ADP (Figure 3). The results of the ctin inding experiments with LP2 AAA MV 1IQ suggest tht the three lysine residues in loop 2 re importnt determinnts of the rte of ssocition with ctin in oth the ADP nd rigor sttes. The 5-fold reduction in the ffinity of LP2AAA MV 1IQ for ctin in the presence of ADP ws solely due to the reduced ctin ssocition rte since the rte of dissocition ws unffected y the muttion. In contrst, the DEEK MV 1IQ construct hd 3-fold fster rte of inding to pyrene ctin in the presence of ADP compred to WT MV 1IQ ut ws similr to WT MV 1IQ in the sence of nucleotide. The rte of inding to ctin in the sence of nucleotide is extremely fst, pproching the diffusion-limited rte of inding (9), nd therefore it is perhps not surprising tht the DEEK MV 1IQ mutnt did not enhnce this rte further. Overll, our results suggest tht the numer of positively chrged mino cids in loop 2 of myosin V correltes with the rte of ssocition with ctin. Our results gree with previous reports tht hve determined the net positive chrge of loop 2 of myosin II controls the rte of inding to ctin (16). In ddition, the removl of the first prt of the unique ctin-inding insert in myosin V does not pper to e criticl for inding to ctin. These results re consistent with the smooth muscle myosin mutnt with 16 mino cids deleted from loop 2, which enhnced its pprent ffinity for ctin without ffecting other steps in

7 Kinetics of Myosin V Loop 2 Mutnts Biochemistry, Vol. 43, No. 9, the myosin ATPse cycle (18). Thus, the size of loop 2 my not e s criticl s the net positive chrge of the loop or perhps the position of the positive chrges, which ply role in modulting the ffinity of myosin for ctin. We exmined the ctin inding kinetics of the LP2AA SmS1, which when exmined in the dimeric HMM construct ws shown to contin very little ctin-ctivted ATPse ctivity or in vitro motility (18). Our results demonstrted 10-fold reduction in the ctin ssocition rte nd 2-3- fold reduction in the mximum rte of LP2AA SmS1 inding to ctin in the presence of ADP compred to WT SmS1. Since the rte of inding to ctin in the presence of ATP is even slower, the LP2AA SmS1 mutnt my indirectly lock ctin ctivtion of phosphte relese y reducing the rte of entry into the strong inding stte. This would explin the single molecule experiments performed with the smooth muscle myosin lysine mutnt, which demonstrted tht the frequency of events oserved ws reduced 20-fold (i.e., the proility of inding strongly to ctin) while the lifetimes of the events were similr to WT SmS1 (the length of time myosin is ound to ctin) (26). The corresponding muttion in myosin V, LP2AAA MV 1IQ, my e much less severe ecuse myosin V hs much fster rte of inding to ctin in the presence of ATP thn myosin II. Overll, these results highlight key kinetic difference etween myosin V nd myosin II in tht the trnsition from wek to strong ctininding conformtion is gretly ccelerted in myosin V. Product Relese Steps. De L Cruz et l. (1999) demonstrted tht the K ATPse of myosin V cn e expressed s function of the following rte nd equilirium constnts. K ATPse ) k +5 /(K 9 k +4 )(K 3 /(K 3 + 1)) In oth the mutnt nd WT MV 1IQ constructs, the V MAX nd ADP relese rte (k +5 ) were quite similr, nd the rte of ATP hydrolysis (k +3 + k -3 ) ws very similr, suggesting tht the equilirium constnt for ATP hydrolysis (K 3 ) ws unffected. Therefore, our results suggest tht the chnges oserved in the K ATPse of the loop 2 mutnts were due to chnges in K 9, k +4, or oth. The results shown in Figure 5 clerly demonstrte tht the mximum rte of phosphte relese (k +4 ) ws similr in the loop 2 mutnt nd WT MV 1IQ constructs, ut the ctin concentrtion dependence (K 9 ) on the rte of phosphte relese ws drmticlly ltered. The clculted K ATPse of the WT nd mutnt MV 1IQ constructs using the ove formul nd our mesured rte nd equilirium constnts is in good greement with the mesured K ATPse in our stedy-stte ATPse experiments, providing further evidence for the vlidity of the formul proposed y De L Cruz nd collegues (1999). The mximum rte of phosphte relese mesured for WT MV 1IQ in the current study ws different thn tht mesured y De L Cruz et l. (9), ut it is uncler wht the cuse of this difference is. However, our results gree within fctor of 2 of the previous report nd do not conflict with the overll conclusion tht phosphte relese is quite rpid in myosin V nd ADP relese is rte-limiting (9). Nonetheless, the current results suggest tht the ffinity for ctin in the M ADP Pi stte (K 9 ) 9 ( 2 µm) is higher thn tht of conventionl myosin II, which is consistent with our previous report tht demonstrted high ctin ffinity in the M ATP stte (11). Interestingly, y reducing the numer of positive chrges in loop 2 of myosin V, such s in LP2AAA MV 1IQ, the ffinity for ctin cn e reduced to vlue tht is more like tht of conventionl myosin II. The high ffinity for ctin in the M ADP Pi stte, which is enhnced y the lrge numer of positively chrged mino cids in loop 2 (net positive chrge of +5), my e criticl for llowing myosin V to move processively long ctin (24). The results from the phosphte relese nd ADP relese experiments suggest tht loop 2 does not ply role in ctin ctivtion of product relese in myosin V. Our results support previous reports tht demonstrted tht loop 2 modultes the ffinity for ctin without significntly ltering the mximum rte of ATPse ctivity (13, 14). Conversely, mutgenesis of loop 2 in Dictyolstelium myosin II demonstrted tht loop 2 is involved in ctivting ADP nd phosphte relese (4, 15-17). In ddition, dding three lysines residues to loop 2 of smooth muscle myosin reduced the rte of ADP relese while not chnging the mximum ATPse rte (27). Thus, it is possile tht the effects of loop 2 on product relese re myosin isoform specific, ut the role of loop 2 in inding to ctin is conserved throughout the myosin superfmily. The positively chrged mino cids in loop 2 my ply role in ctin inding y mediting the initil interction etween ctin nd myosin. The positively chrged mino cids in loop 2 likely interct with the highly negtively chrged N-terminus of ctin. Indeed, mutting or deleting severl cidic residues in the N-terminl region of ctin drmticlly chnged myosin inding ffinity ut did not completely lock ctin ctivtion of myosin ATPse ctivity (28). Thus, interctions etween loop 2 nd the N-terminus of ctin my provide the initil electrosttic steering etween ctin nd myosin, which llows the formtion of other hydrophoic nd ionic interctions to generte the strongly ound ctomyosin complex. Our results suggest the primry role of loop 2 in myosin V is to modulte the wek inding ffinity. Since myosin V moves processively long ctin tking 36 nm steps, its highly chrged loop 2 my e importnt to llow one hed to find the next ctin inding site efore the other hed detches. In conclusion, we hve reveled tht loop 2 of myosin V plys n importnt role in llowing myosin V to ind ctin with reltively high ffinity in the wek inding sttes. The net positive chrge nd not the size of loop 2 ppers to e criticl for myosin V s high-ffinity ctin inding. In contrst to studies on myosin II, loop 2 does not ply direct role in ctin ctivtion of product relese in myosin V. Thus, the lrge nd highly chrged loop 2 of myosin V hs kineticlly tuned myosin V to increse its ffinity for ctin, which my e importnt for its ility to move processively long ctin nd function s n orgnelle trnsporter. ACKNOWLEDGMENT We thnk Steven Rosenfeld for generously providing the phosphte-inding protein. We lso thnk E. Michel Ostp nd Crl Morris for their comments nd creful reding of this mnuscript. We lso recognize the excellent technicl work of Corey Bldcchino nd Jocelyn Nolt. REFERENCES 1. Sellers, J. R. (1999) Myosins, 2nd edition, Protein Profile, Oxford University Press, Oxford, U.K.

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