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1 SUPPLEMENTARY INFORMATION doi:138/nture123 Supplementry Discussion Chrcteriztion of ABI-PP s sustrte of efflux trnsporters. Among efflux trnsporter inhiitors, oligopeptide derivtives such s PAβN re exported y RND type multidrug efflux trnsporters 21. Here we determined whether ABI-PP is exported y efflux trnsporters. The direct mesurement of the efflux trnsport medited y the purified RND trnsporter complex is impossile ecuse no one hs succeeded in reconstituting ctive RND triprtite complexes tht penetrte inner nd outer memrnes. In this study, we used intct crb-deficient E. coli cells in which AcrB ws expressed from plsmid nd compred these cells with host cells hving no AcrB. Most efflux trnsporter sustrtes enter into cells primrily y simple diffusion through the lipid mtrix of the cell memrne due to their mphiphilic nture. Therefore, when drugs were dded to the medium contining E. coli cells without the efflux trnsporter nd the concentrtion of drugs in the medium ws monitored, the concentrtion grdully decresed due to the ccumultion of the drugs in the cells. The concentrtions of drugs in the medium were mesured y LC-MS/MS. As shown in Supplementry Fig. S2, the doxoruicin concentrtion in the medium contining crb-deficient E. coli cells exhiited time-dependent decrese, wheres it showed lmost no chnge during 6 min in the medium contining crb-expressing cells ecuse the ctive efflux of doxoruicin prevents ccumultion in cells (Supplementry Fig. S2). This result verifies tht doxoruicin is good sustrte for export y AcrAB-TolC. In contrst, in the cse of crenicillin, the concentrtion in the medium grdully decresed regrdless of whether AcrB ws expressed (Supplementry Fig. S2). This oservtion verifies tht crenicillin is not exported y AcrAB-TolC. Supplementry Fig. S2c shows tht the ABI-PP concentrtion in the medium decresed regrdless of whether AcrB ws expressed, s oserved for crenicillin, indicting tht ABI-PP is hrdly exported y AcrAB-TolC t lest in the concentrtions used s inhiitors. Binding of ABI-PP to efflux trnsporters. The inding of ABI-PP to efflux trnsporters ws nlyzed using purified trnsporter proteins ecuse the ctive triprtite complex is not ville. The otined AcrB, MexB nd MexY proteins were very pure nd hd distinct moleculr weights on the SDS PAGE gel (Supplementry Fig. S3). The inding prmeters were mesured y isotherml titrtion clorimetry (ITC). As shown in Supplementry Fig. S3, AcrB nd MexB showed sturtion inding curves, indicting tht ABI-PP inds to these proteins in specific mnner. The Kd vlues for ABI-PP to AcrB nd MexB were 1.15 μm nd 3.57μM, respectively. The molr rtio of ABI-PP to AcrB/MexB trimers ws 1:1. In contrst, MexY showed no sturtion of ABI-PP inding except for sl non-specific inding. The inding energies clculted using Glide (Schrödinger) were -5.6, -5.2 nd kcl/mol for the inding of minocycline, doxoruicin nd ABI-PP, respectively, with AcrB nd were -5.5 nd 1

2 RESEARCH SUPPLEMENTARY INFORMATION kcl/mol for the inding of DDM nd ABI-PP, respectively, with MexB, indicting tht ABI-PP inds much more strongly thn the norml sustrtes. Competitive inding of ABI-PP with other drugs. The competitive inding of ABI-PP with minocycline nd rifmpicin to purified AcrB proteins ws mesured y LC-MS/MS (Supplementry Fig. S4). The dvntge of this nlysis is its ility to distinguish ABI-PP nd drugs in the sme medium. Although the correct mounts of ound drugs/inhiitors re hrdly determined y this method ecuse of the dissocition during gel filtrtion nd, on the contrry, the nonspecific inding of mphiphilic drugs/inhiitors to memrne proteins, we otined highly reproducile results when the experimentl conditions were kept constnt. As shown in Supplementry Fig. S4, protein-ound drugs/inhiitors re clerly detected in the void protein frctions. Wheres only out 2 % of drugs (minocycline) ound with AcrB, more thn 8 % of inhiitors (ABI-PP) remined to e ound with AcrB even fter gel filtrtion, indicting strong inding of ABI-PP to AcrB. The elution of free minocycline ws significntly delyed fter totl column volume proly due to the dhesion of drugs to gel. The dt in Supplementry Fig. S4 correspond to the men vlues nd stndrd devitions of 3 independent experiments. The dt were normlized sed on the mount of ound drugs without the competitors. Although there ws lrge mount of non-specific inding, most likely similr in mgnitude (~.5) to the specific inding, the results convey the importnt fcts, tht is, tht ABI-PP inhiits the inding of distl-inding drug minocycline, ut does not inhiit the inding of proximl-inding drug, rifmpicin. As reported in our pervious pper 13, minocycline inds to the distl pocket of the inding monomer, wheres rifmpicin inds to the proximl pocket of the ccess monomer. These two drugs do not compete with ech other for inding sites, nd we cn determine the structure of the AcrB trimer ound simultneously to minocycline in the inding monomer nd rifmpicin in the ccess monomer 13. Nevertheless, the trnsport of the distl-inding drug is competitively inhiited y the proximl-inding drug, indicting tht the trnsport pthwys for oth drugs re the sme 13. The dt in Supplementry Fig. S4 indicte tht ABI-PP is distl-inding inhiitor. Another importnt result shown in Supplementry Fig. S4 is tht the mount of ound ABI-PP ws not sustntilly chnged y either minocycline or rifmpicin, indicting tht the ffinity of ABI-PP for the protein is higher thn the ffinities of the trnsported sustrtes. The tight inding of ABI-PP is the sis of its inhiitory ctivity. Chrcteriztion of ABI-PP s n exporter inhiitor to potentite the ctivities of vrious ntiiotics. Supplementry Fig. S5 shows the potentition of vrious ntiiotics y ABI-PP. Ech pnel indictes the growth of crb/tolc-deficient E. coli cells expressing MexAB-OprM or MexXY-OprM from plsmids. The upper pnels indicte the growth of MexAB-OprM-expressing cells nd the lower pnels indicte the growth of MexXY-OprM-expressing cells. Blck lines nd 2

3 SUPPLEMENTARY INFORMATION RESEARCH colored lines indicte the growth in the sence nd presence of ABI-PP (32 μg/ml), respectively, in ddition to the ntiiotics stted. ABI-PP itself hs no ntiiotic ctivity (Supplementry Fig. S1). As shown in Supplementry Fig. S5, oth MexB- nd MexY-expressing E. coli cells cn normlly grow in the presence of the indicted mounts of ntiiotics without ABI-PP. The upper pnels show tht, in the presence of 32μg/ml ABI-PP, the growth of MexB-expressing cells ws completely inhiited or lrgely decresed. In contrst, the lower pnels indicte tht the growth of MexY-expressing cells ws lmost not ffected y the presence of ABI-PP. In the cse of AcrB-expressing cells, the ctivities of the ntiiotics were potentited y ABI-PP similr to the cse of MexB-expressing cells (dt not shown). These results indicte tht ABI-PP is n AcrB/MexB-specific inhiitor nd does not inhiit MexY. Crediility of the moleculr model of the mino cid side chins round ABI-PP in AcrB nd MexB. The electron density mps (2Fo-Fc; cyn mesh) corresponding to the mino cid residues round the ound ABI-PP were dded to Fig. 1c nd Fig. 2c (Supplementry Fig. S6). The 2Fo-Fc mps for the ABI-PP-inding pits of AcrB nd MexB re sufficient to ensure tht the moleculr model of the mino cid side chins is correct. Crystl structure of drug-free MexB. The drug-free MexB structure ws solved t 2.7 Å (Supplementry Fig. S7). A ound DDM molecule ws detected in the distl pocket of the inding monomer nd n dditionl 8 DDM molecules were ttched to the trnsmemrne domin (Supplementry Fig. S7 nd ). In ddition, the centrl hole of the trnsmemrne domin ws filled with electron densities tht most likely correspond the cyl chins of memrne phospholipids (Supplementry Fig. S7c nd d). Becuse we used n E. coli strin tht possesses n intrinsic crb gene s the expression host, AcrB contmintion my e prolem (Veesler D. et l. Act Cryst. 64, (28)). To eliminte the possiility of AcrB contmintion, we identified the MexB protein in the crystl y SDS PAGE (Supplementry Fig. S3). In ddition, when moleculr refinement ws performed using the MexB model truncted round F175 (in AcrB, this position is occupied y vline), the electron density corresponding to the F175 side chin ws clerly detected, indicting tht the structure is certinly MexB nd not AcrB (Supplementry Fig. S7e). DDM inding site in MexB. In the electron density mp of the drug-free MexB, there is n excess density in the distl inding pocket, the shpe of which exctly mtches tht of detergent molecule (DDM) (Supplementry Fig. S8). The cyl chin of the DDM molecule is inserted into the hydrophoic pit nd prtly overlps with the ABI-PP inding site. This electron density is significntly different from tht of the ABI-PP molecule (Supplementry Fig. S8). The structure of ABI-PP-ound MexB exhiits n electron density corresponding to ABI-PP in this position; this 3

4 RESEARCH SUPPLEMENTARY INFORMATION density mtches exctly expected density for the ABI-PP molecule nd is clerly different from tht of DDM molecule. Therefore, it is cler tht ABI-PP inds to MexB replcing the DDM molecule. The MexB F178W mutnt is still sensitive to ABI-PP. Supplementry Fig. S11 shows the ility of ABI-PP to potentite erythromycin nd inhiit doxoruicin efflux in MexB nd MexB F178W-expressing E.coli cells. There ws no difference etween MexB nd MexB F178W-expressing strins with respect to the effect of ABI-PP, indicting tht ABI-PP is still n efficient inhiitor of MexB F178W. The doxoruicin concentrtion grdully decresed even in the sence of ABI-PP, indicting tht MexB nd MexB F178W do not completely prevent the intrcellulr ccumultion of doxoruicin ecuse this drug is not good sustrte for MexB. When ABI-PP ws dded, the rte t which the doxoruicin concentrtion decresed ws significntly fster thn the rte in the sence of ABI-PP, clerly indicting tht ABI-PP inhiits oth MexB nd MexB F178W. Crystl structures of AcrB F178W, MexB F178W nd ABI-ound MexB F178W. To determine why AcrB F178W is not inhiited y ABI-PP even though MexB F178W is still sensitive to ABI-PP, we solved the crystl structures of drug-free AcrB F178W, drug-free MexB F178W nd ABI-PP-ound MexB F178W t 3.6 Å, 3.3 Å nd 3. Å resolutions, respectively. The overll structures did not differ from their wild-type structures. Supplementry Fig. S12 shows close-up view of the hydrophoic pits round W178. These three figures re viewed from the sme direction. An ABI-PP molecule is certinly present in the hydrophoic pit of MexB F178W (Supplementry Fig. S12c). The electron density corresponding to the mino cid side chins of ABI-PP-ound MexB F178W ws significntly clerer thn those of the drug-free structures, indicting tht ABI-PP inding fixes the positions of the surrounding residues. In pnels nd, the electron density corresponding to W178 ws clerly detected ut ws somewht poorer thn tht in the ABI-PP-ound structure. In AcrB F178W, the indolyl ring protrudes to nrrow pit, wheres in MexB F178W, this ring does not protrude ut is prllel to the groove of the pit ecuse there is ound DDM molecule present. The ABI-PP-ound structure ws not significntly different from the ABI-PP-free structure with respect to the conformtions of W178 nd the surrounding residues. Structurl evlution to determine why AcrB F178W nd MexY do not ind ABI-PP. Supplementry Fig. S13 shows comprison of the ABI-PP-ound structure of MexB F178W nd the imginry structures of ABI-PP-ound AcrB F178W nd MexY. Pnel shows the close-up structure of the hydrophoic pit of wild-type AcrB in which F178 ws replced y tryptophn. The orienttion of the indolyl ring ws rotted to llow ABI-PP inding. However, in this conformtion, the indolyl side chin overlps with the electron density of the surrounding residues especilly tht 4

5 SUPPLEMENTARY INFORMATION RESEARCH of V139. In contrst, in MexB F178W shown in pnel, the indolyl side chin of W178 fits into the wll composed of the surrounding residues. As for the MexY homology model, the sitution is similr to tht of AcrB F178W ecuse of the steric hindrnce with I138. To remove the steric hindrnce, we replced V139 nd I138 y lnine. The resulting mutnts, AcrB F178W/V139A nd MexY I138A were ctully inhiited y ABI-PP (Supplementry Fig. S14). Thus, the inhiitor s specificity is determined y the smll differences of the spce in the inhiitor inding pit round F178 (AcrB/MexB) nd W177 (MexY). 5

6 RESEARCH SUPPLEMENTARY INFORMATION Supplementry Figure S1. Chemicl structure of thepyridopyrimidine derivtive,abi-pp c 12 Drug undnce rtio in roth (%) DOX DcrB ΔcrB DOX pcrb Time (m) Drug undnce rtio in roth (%) CAR DcrB ΔcrB CAR pcrb Time (m) Drug undnce rtio in roth (%) ABI-PP D13-91 DcrB ΔcrB ABI-PP D13-91 pcrb pcrb Time (m) Supplementry Figure S2. Drug efflux from E. coli cells mesured s the prevention of the time-dependent decrese in the drug concentrtion in the medium s result of drug ccumultion in the cells. Drugs (28.6 μm) were mixed with crb-deficient cells (ornge line) nd the cells expressing crb from the plsmid (red line), respectively. At the indicted time intervls, the mount of drugs in the medium ws mesured y LC-MS/MS using Wters Acquity UPLC system (Wters Corportion, Milford, MA, USA) nd Wters Quttro Premier TM XE triple qudruple mss spectrometer (Micromss MS Technologies, Mnchester, UK) equipped with n ESI interfce. The drug undnce rtio (verticl xis) ws clculted y dividing the pek intensities from the mss spectrum of the drug t the indicted times y the pek intensity t time zero.. Doxoruicin, good sustrte for AcrAB-TolC.. Crenicillin which is not sustrte of AcrAB-TolC. c. ABI-PP, n inhiitor of AcrAB-TolC. The dt correspond to the men vlues of 3 independent experiments. The error rs correspond to the stndrd devition. 6

7 SUPPLEMENTARY INFORMATION RESEARCH KD mrker MexY (purified protein) MexB (crystl) AcrB (crystl) AcrB MexB MexY MexY 82 MexB AcrB 68 Supplementry Figure S3. Purifiction of AcrB, MexB nd MexY nd the inding of ABI-PPto these purified trnsporter proteins.. SDS-PAGE of the purified proteins (MexY) nd proteins from crystls (AcrB nd MexB) stined with Coomssie Brillint Blue.. Isotherml titrtion clorimetry (ITC) nlysis of the inding of ABI-PP to purified AcrB, MexB nd MexY proteins. The experiments were performed with Nno ITC-LV system (TA Instruments, Inc., New Cstle, DE) t 25 ºC. The rection cell ws filled with 7 μm purified trnsporter proteins (s trimer) nd 2 injections of 2.46 μl liquots of 4 μm ABI-PP dissolved in the sme uffer (2 mm Nphosphte, ph 6.2, % DDS for AcrB or.5 % DDM for MexB nd MexY) were performed t 12 sec intervls. The het due to the inding rection is plotted versus the ABI-PP/trnsporter trimer molr rtio. The dt were fitted using the softwre Nno Anlyze

8 RESEARCH SUPPLEMENTARY INFORMATION Supplementry Figure S4. Competitive inding of ABI-PP with drugs to purified AcrB. ABI-PP nd drugs were mixed with purified AcrB protein (25 μm, 25 μm nd 2μM, respectively) in 2mM sodium phosphte (ph 6.2), % DDS. After gel filtrtion (HiTrp Deslting, GE Helthcre), compounds were extrcted from void frction (frction No. 3) y 5% cetonitrile nd the mount of compounds were mesured y LC-MS/MS (see Fig. S1). All experiments were performed t room temperture.. Elution profile of gel filtrtion. Column volume is 5 ml nd ech frction is.6 ml. AcrB proteins were detected y SDS-PAGE (stined with CBB). ABI-PP nd minocycline were detected y the sornce t 42 nm nd 35 nm, respectively. Asornce due to proteins ws sutrcted from the sornce in frctions 3 nd 4. Red nd lue colors indicte the dt with AcrB nd without AcrB, respectively.. Amount of ound drugs extrcted from frction 3, normlized y the mount of ound drugs without competitors (other drugs or inhiitors). The dt correspond to the men vlues of 3 independent experiments. The error rs correspond to the stndrd devition (Student s t-test, *P<.5). MN, minocycline; RIF, rifmpicin; PP, ABI-PP. 8

9 SUPPLEMENTARY INFORMATION RESEARCH A6 nm A6 nm OXA MINO c R6G d EBR e CIP f PP (μg/ml) PP (μg/ml) PP PP (μg/ml) PP (μg/ml) PP (μg/ml) PP (μg/ml) g h i j k l PP (μg/ml) PP (μg/ml) (μg/ml) PP (μg/ml) LEV.7.6 PP (μg/ml) PP (μg/ml) Supplementry Figure S5. The inhiitory effect of ABI-PP on cell growth in the presence of vrious ntiiotics. ABI-PP is shown s PP in the pnels. -f. The growth of E. coli cells expressing wild-type MexB in the presence of 124 μg/ml oxcillin (), 25 μg/ml minocycline (), 2 μg/ml rhodmine 6G (c), 4 μg/ml ethidium romide (d),.8 μg/ml ciprofloxcin (e) or.15 μg/ml levofloxcin (f) with or without ABI-PP. g-l. The growth of E. coli cells expressing wild-type MexY in the presence of 512 μg/ml oxcillin (g), 25 μg/ml minocycline (h), 1 μg/ml rhodmine 6G (i), 8 μg/ml ethidium romide (j),.8 μg/ml ciprofloxcin (k) or.3 μg/ml levofloxcin (l) with or without ABI-PP. These experiments were repeted t lest three times. 9

10 RESEARCH SUPPLEMENTARY INFORMATION N274 N274 S18 R62 S18 R62 L177 I277 L177 I277 Q176 F136 F178 V139 F615 A279 V612 F61 F628 Q176 F136 F178 V139 F615 A279 V612 F61 F628 P326 M573 P326 M573 Y327 L668 Y327 L668 V571 V571 R128 S18 D274 R128 S18 D274 Q46 R62 Q46 R62 V177 K151 I277 F615 V177 K151 I277 F615 Q176 F136 F178 V139 F61 F628 V612 A279 Q176 F136 F178 V139 A279 V612 F61 F628 F573 F573 Y327 L627 M63 Y327 L627 M63 V571 V571 Supplementry Figure S6. Electron densities round the ABI-PP inding sites of AcrB () nd MexB (). The ound ABI-PP nd the mino cid ide chins re the sme s in Fig. 1c nd Fig. 2c. The 2Fo-Fc mps contoured t 1. σ corresponding to mino cid residues re represented y cyn mesh. The Fo(drug inding) Fo(drug free) mp corresponding to ABI-PPcontoured t 3. σ is represented y n ornge mesh in. The Fo-Fc omit mp corresponding to ABI-PP contoured t 3. σ is represented y n ornge mesh in. 1

11 SUPPLEMENTARY INFORMATION RESEARCH c e d F175 9 Supplementry Figure S7. Crystl structure of the drug-free MexB.. The entire structure of drug-free MexB. Molecules of the detergent, n-dodecyl-β-d-mltoside (DDM), re contined in this structure (purple). Eight DDM molecules re ound to the trnsmemrne domin of MexB trimer, nd 1 is ound to the distl pocket of the inding monomer.. Cutwy view of the trnsmemrne domin from the cytoplsmic side. c. Close-up view of the centrl hole of drug-free MexB in the sme view s. The 2Fo-Fc mp is represented y green mesh contoured t 1. σ (c nd d). The hole is filled y electron densities corresponding to cyl chins tht most likely elong to memrne phospholipids. d. Cutwy view in verticl with the memrne plne. e. Electron density mp of the truncted prt of MexB. Becuse we used n E. coli strin possessing n intrinsic crb s the expression host, in ddition to the SDS-PAGE nlysis (Supplementry Figure S2), we verified tht the protein forming the crystl ws ctully MexB nd not AcrB. All mino cids within 1 Å of F175 (the corresponding position in AcrB is vline) were deleted from the MexB model. Moleculr replcement ws then performed using this truncted MexB s serch model, nd the model ws susequently refined using Refmc (rigid-ody refinement, restrint refinement). The Fo-Fc mp nd the 2Fo-Fc mp re represented y red mesh (contoured t 3.σ) nd cyn mesh (contoured t 2.σ), respectively. The truncted MexB is shown in yellow sticks, nd the MexB model efore trunction is superposed nd represented y green lines. c Supplementry Figure S8. Electron density mp (2Fo-Fc) contoured t 1. σ round the DDM inding site in the distl pocket of drug-free MexB. Electron densities derived from the protein nd the ound compound re shown in ornge nd lue, respectively.. Close-up stereo view of the DDM inding site with ound DDM molecule.. MexB-ound ABI-PP overlps with the DDM inding site. c. MexB-ound DDM overlps with the ABI-PP inding site. W W W. N A T U R E. C O M / N A T U R E 1 1

12 RESEARCH SUPPLEMENTARY INFORMATION mrker (Dr. western) pbad33 pbadcrb pbadcrb F178W pmmb67he pmexab his M pmexab(f178w) his M pmexxy his M pmexxy(w177f) his M mrker (Precision Plus) KD KD MexY MexB AcrB Supplementry Figure S9. Expression of the efflux trnsporter proteins from plsmids. Exponentil phse cells contining plsmids encoding mutnt or wild-type trnsporter were disrupted y soniction nd the memrne frctions were collected y ultrcentrifugtion. The memrne frctions were seprted y SDS- PAGE nd the trnsporters were visulized y western lot nlysis using monoclonl nti-polyhistidine ntiodies. An equl mount of memrne proteins (2 μg) ws loded in ech lne. Wild-type nd mutnt AcrB proteins encoded y pbad-derived plsmids were expressed in n crb-deficient E. coli strin. MexAB- OprM nd MexXY-OprM encoded y pmmb-derived plsmids were expressed in n crb/tolc-deficient E. coli strin. 12

13 SUPPLEMENTARY INFORMATION RESEARCH A6 nm PP PP8 PP16 (μg/ml) PP (μg/ml) PP8 PP c A6 nm PP (μg/ml) PP8 PP d PP (μg/ml) PP8 PP e A6 nm PP (μg/ml) PP8 PP f PP (μg/ml) PP8 PP Supplementry Figure S1. Growth of crb-deficient (, ) nd crb/tolc-deficient (c-f) E. coli cells hroring plsmids encoding efflux trnsporter genes in the presence of vrious concentrtions of ABI-PP without ntiiotic. ABI-PP is shown s PP in the pnels.. wildtype crb,. crb F178W, c. wild-type mexb, d. mexb F178W, e. wild-type mexy, f. mexy W177F. As for mexb nd mexy, mexa-oprm nd mexx-oprm were lso expressed from the sme plsmid, respectively. These tests were repeted t lest three times. 13

14 RESEARCH SUPPLEMENTARY INFORMATION A6 nm PP (μg/ml) PP5 PP.5 PP1 PP PP (μg/ml) PP5 PP.5 PP1 PP2 c Fluorescence intensity (.u.) PP- PP PP- PP Time (m) d Time (m) Supplementry Figure S11. The inhiitory effect of ABI-PP on MexB. ABI-PP is shown s PP in the pnels. -. Growth of crb/tolc-deficient E. coli cells hroring plsmid encoding wildtype mexa/mexb/oprm () or mexa/mexb F178W/oprM () in the presence of 2 μg/ml erythromycin nd vrious concentrtions of ABI-PP. The erythromycin susceptiility experiments were repeted t lest 3 times. c-d. The effect of ABI-PP (28.6 μm) on the quenching of doxoruicin fluorescence in crb/tolc-deficient E. coli cells hroring plsmid encoding wildtype mexa/mexb/oprm (c) or mexa/mexb F178W/oprM (d). The dt correspond to the men vlues of 3 independent experiments. The error rs correspond to the stndrd devition. 14

15 SUPPLEMENTARY INFORMATION RESEARCH c Supplementry Figure S12. Close-up views of the hydrophoic pits in the inding monomers of F178W mutnts with or without ABI-PP. Drug-free AcrB F178W (, green), drug-free MexB F178W (, lue) nd ABI-PP (yellow)-ound MexB F178W (c, red) re depicted using the stick model. The indolyl side chins of W178 in AcrB F178W nd MexB F178W re shown in white. The 2Fo-Fc electron density mps re shown s ornge meshes contoured t.8,.8, nd 1. σ, respectively. c W178 W178 W177 V139 I138 Supplementry Figure S13. Evlution of the puttive ABI-PP-ound structures.. Close up view of the hydrophoic pit of ABI-PPound MexB F178W mutnt.. The sme view s in of the ABI-PP-ound wild-type AcrB in which F178 is replced y Trp. The orienttion of W178 is rotted s in. The pink color indictes the surfce of V139, which overlps with the indolyl ring of W178. c. The sme view s in nd for the MexY homology model. ABI-PP is plced s in nd the orienttion of W177 is rotted s in. The pink color indictes the surfce of I138, which overlps with W

16 RESEARCH SUPPLEMENTARY INFORMATION (μg/ml) (μg/ml) A6 nm Supplementry Figure S14. The inhiitory effect of ABI-PP on AcrB F178W/V139A nd MexY I138A. ABI-PP is shown s PP in the pnels. Growth of crb-deficient () nd crb/tolcdeficient () E. coli cells hroring plsmids encoding crb F178W/V139A () or mexx/mexy I138A/oprM () in the presence of 6 μg/ml () or 16 μg/ml () erythromycin nd vrious concentrtions of ABI-PP. These experiments were performed t lest three times. 16

17 SUPPLEMENTARY INFORMATION RESEARCH Supplementry Tle 1. Dt collection nd refinement sttistics. Dt Collection AcrB + ABI-PP MexB + ABI-PP MexB AcrB F178W MexB F178W MexB F178W + ABI-PP Light source PF-AR NW12A SPring8 BL44XU SPring8 BL44XU SPring8 BL44XU SPring8 BL44XU SPring8 BL44XU Spce group C2 P1 P1 C2 P1 P1 Wvelength (Å) Cell dimensions,, c (Å) , , , 137.3, , , , , , , , , α, β, γ ( ) 9., 97.83, , 68.93, , 69.49, , 97.73, , 69.97, , 69.29, Resolution (Å) 5-3.5( ) ( ) ( ) 5-3.6( ) 5-3.3( ) 5-3.(3.5-3.) R merge 6. (62.2) 9.1 (63.) 4.7 (55.2) 7.5(63.7) 7.2(61.2) 5.2(56.) I/σI 21.9 (1.6) 2 (2.6) 37.8 (2.4) 3.7(3.1) 21.3(2.3) 26.3(2.6) Completeness (%) 97.7 (97.8) 98.8 (98.8) 97.5 (96.9) 99.6(1.) 99.2(98.8) 99.(98.9) Redundncy 3.5 (3.4) 3.9 (3.9) 3.8 (3.7) 7.6(7.8) 3.9(4.) 4.(4.) Refinement Resolution (Å) No. reflections 36,35 63,42 918, , , ,24 R work /R free 23.1/ / / / / /3. No. toms Protein 23,55 46,678 45,745 23,331 46,78 46,751 Lignd/ion Wter 7 7 B-fctors Protein Lignd/ion Wter R.m.s. devitions Bond lengths (Å) Bond.ngles ( ) *Highest resolution shell is shown in prenthesis. Supplementry Tle S2. Drug resistnce of E. coli cells expressing efflux trnsporter genes from the plsmids. Strin Minimum inhiitory concentrtion, MIC (µg/ml) OXA EM DXR MINO R6G EBR Wild type > >512 ΔcrB ΔcrB/pBAD ΔcrB/pBADcrB > ΔcrB/pBADcrB F178W > ΔcrBΔtolC ΔcrBΔtolC/pMMB67HE* ΔcrBΔtolC/pMexAB his M ΔcrBΔtolC/pMexAB(F178W) his M ΔcrBΔtolC/pMexXY his M ΔcrBΔtolC/pMexXY(W177F) his M OXA, oxcillin; EM, erythromycin; DXR, doxoruicin; MINO, minocycline; R6G, rhodmine 6G; EBR, ethidium romide. Vlues in old indicte the significnt increse compred with the corresponding prentl strins. MIC determintions were repeted t lest 3 times. *Oxcillin resistnce of the trnsformed cells incresed ecuse the resistnce mrker of pmmb67he is sulenicillin r, which provides resistnce ginst β-lctms including oxcillin. 17