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Structure and activity of lipid bilayer within amembrane-protein transporterWeihua Qiua,b,1, Ziao Fuc,1, Guoyan G. Xua, Robert A. Grassuccid, Yan Zhanga, Joachim Frankd,e,2,Wayne A. Hendricksond,f,g,2, and Youzhong Guoa,b,2

aDepartment of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23298; bInstitute for Structural Biology, Drug Discovery andDevelopment, Virginia Commonwealth University, Richmond, VA 23219; cIntegrated Program in Cellular, Molecular, and Biomedical Studies, ColumbiaUniversity, New York, NY 10032; dDepartment of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032; eDepartment ofBiological Sciences, Columbia University, New York, NY 10027; fDepartment of Physiology and Cellular Biophysics, Columbia University, New York,NY 10032; and gNew York Structural Biology Center, New York, NY 10027

Contributed by Wayne A. Hendrickson, October 15, 2018 (sent for review July 20, 2018; reviewed by Yifan Cheng and Michael C. Wiener)

Membrane proteins function in native cell membranes, but extrac-tion into isolated particles is needed for many biochemical andstructural analyses. Commonly used detergent-extraction meth-ods destroy naturally associated lipid bilayers. Here, we devised adetergent-free method for preparing cell-membrane nanopar-ticles to study the multidrug exporter AcrB, by cryo-EM at 3.2-Åresolution. We discovered a remarkably well-organized lipid-bilayer structure associated with transmembrane domains of theAcrB trimer. This bilayer patch comprises 24 lipid molecules; innerleaflet chains are packed in a hexagonal array, whereas the outerleaflet has highly irregular but ordered packing. Protein side chainsinteract with both leaflets and participate in the hexagonal pattern.We suggest that the lipid bilayer supports and harmonizes peristal-tic motions through AcrB trimers. In AcrB D407A, a putative proton-relay mutant, lipid bilayer buttresses protein interactions lost incrystal structures after detergent-solubilization. Our detergent-freesystem preserves lipid–protein interactions for visualization andshould be broadly applicable.

AcrB | cryo-EM | nanoparticle | phospholipid | styrene maleic acidcopolymer

Cell membranes and their constituent proteins are crucial forliving organisms, and great efforts have been made to un-

derstand the structures of cell-membrane systems (1–8). De-tergent solubilization has dominated membrane-protein studies(9, 10); however, detergents have significant drawbacks becausethey destroy cell membranes and remove protein-associated lipidmolecules (11, 12). Protein–lipid interactions play crucial rolesfor membrane proteins; for example, activity of mitochondrialrespiratory complex I extracted with detergents suffers exten-sively from the depletion of lipid components (13, 14). Theimportance of the protein–lipid interactions in biology andmedicine fosters the need for procedures that preserve lipidswhile extracting proteins from membranes.Membrane-active polymers such as styrene maleic acid (SMA)

copolymer, diisobutylene maleic acid copolymer, and others havebeen shown to be useful in membrane studies (15–17). Extrac-tion of membrane proteins into SMA lipoprotein particles(SMALPs) was demonstrated first from proteoliposomes (16),but similar procedures also permit direct solubilization fromcell membranes, never employing detergents (18). SMA co-polymer has emerged as an alternative to traditional detergentsfor membrane-protein research (18–21), including use in struc-tural analysis (22, 23). A recent cryo-EM analysis of SMA-extracted AcrB reached a resolution limit of 8.8 Å (24).AcrB is an archetypal resistance-nodulation-division multidrug

exporter from the inner cell membrane of gram-negative bacte-ria. Crystal structures of AcrB from Escherichia coli were firstreported as symmetric trimers (25–27) and later as asymmetrictrimers (28, 29). Because of its biological and biomedical im-portance, AcrB has been investigated extensively and many AcrBstructures have been reported, having resolutions as high as

1.9 Å (30). Nevertheless, the mechanism of active transport is stillfar from clear, in part because crucial structural information re-garding protein–lipid interaction is missing (31). The AcrB trimerhas a central cavity between transmembrane (TM) domains of thethree protomers, where a portion of lipid bilayer may exist (26).Although detergent molecules and some alkane chains have beenidentified, organized lipid structure has eluded detection in thecentral cavity or elsewhere.We have developed a native cell-membrane nanoparticles

system based on the previously reported SMALP method (18)for high-resolution structure determination using single-particlecryo-EM. Here, we report our discovery of a high-resolutionstructure of lipid bilayer in extracted nanoparticles of AcrB.The structure of the lipid bilayer and its interaction with AcrBprovide us with important insights both for understanding the

Significance

Membrane proteins function naturally as imbedded in the lipidbilayers of cell membranes, but isolation into homogeneousand soluble preparations is needed for many biochemicalstudies. Detergents, which are used traditionally to extract andpurify membrane proteins from cells, also remove mostprotein-associated lipid molecules as they disrupt the mem-branes. We have devised a detergent-free system to preparenative cell-membrane nanoparticles for biochemical analysis. Inapplication to the membrane transporter AcrB, we demon-strate that these detergent-free nanoparticles are suitable forcryo-EM imaging at high resolution and that the natural lipid-bilayer structure so preserved is important for the functionalintegrity of AcrB. This nanoparticle system should be broadlyapplicable in membrane-protein research.

Author contributions: W.Q. and Y.G. designed research; W.Q., Z.F., G.G.X., and Y.G. per-formed research; Y.G. supervised all of the work; J.F. supervised the EM experiments;R.A.G. set up and maintained EM facilities; Y.Z. gave advice on chemical synthesis;W.Q., G.G.X., Y.Z., and Y.G. contributed new reagents/analytic tools; W.Q., Z.F., W.A.H.,and Y.G. analyzed data; W.A.H. gave advice on structure analysis; W.Q., Z.F., G.G.X.,R.A.G., Y.Z., J.F., W.A.H., and Y.G. wrote the paper.

Reviewers: Y.C., University of California, San Francisco; and M.C.W., University of Virginia.

The authors declare no conflict of interest.

Published under the PNAS license.

Data deposition: Three-dimensional density maps and atomic models have been depos-ited in the Electron Microscopy Data Bank, www.ebi.ac.uk/pdbe/emdb [EMDB entry nos.EMD-7074 (www.ebi.ac.uk/pdbe/entry/emdb/EMD-7074; wild-type AcrB and lipid bilayer)and EMD-7609 (www.ebi.ac.uk/pdbe/entry/emdb/EMD-7609; AcrB D407A mutant andlipid bilayer)], and the Protein Data Bank, www.wwpdb.org [PDB ID codes 6BAJ (wild-type AcrB and lipid bilayer) and 6CSX (AcrB D407A mutant and lipid bilayer)].1W.Q. and Z.F. contributed equally to this work.2To whom correspondence may be addressed. Email: [email protected], [email protected], or [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1812526115/-/DCSupplemental.

Published online December 3, 2018.

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active mechanism of this transporter and for understandingprotein–lipid interactions in cell membranes generally.

Results and DiscussionLipid Bilayer Ordering in Native Cell-Membrane Particles of AcrB. Weprepared native cell-membrane nanoparticles of E. coli AcrBusing membrane-active SMA polymers. The nanoparticles werepurified by single-step Ni-affinity chromatography, applied directlyto grids, and vitrified for single-particle cryo-EM analysis. A 3Dreconstruction with C1 symmetry achieved a final density map of3.2-Å resolution (Fig. 1 A and B and SI Appendix, Fig. S1). Weinitially tried to reconstruct the 3D EM map in C3 symmetry;however, that density map was fragmented, especially so in theTM region, and we could not see lipid-bilayer structure in thecentral cavity. The C1 reconstruction was fitted by an asymmetricAcrB trimer (Fig. 1C and SI Appendix, Fig. S2), where each pro-tomer exists in a distinct state (L for loose, binding-ready; T fortight, substrate-bound; and O for open, substrate release) as in theasymmetric crystal structures (28, 29, 32), but here in this cryo-EMstructure with differences from corresponding subunits in thecrystal structures [r.m.s.d. on Cα positions of 1.9 Å (L), 1.2 Å (T),and 1.0 Å (O) vs. PDB ID code 4U8Y (32)].There is a distinct lipid belt around the TM region and a patch

of ordered lipid bilayer in the lipid cavity of the AcrB trimer (Fig.1). The TM density covered by the protein model has a diameterof ∼9 nm, whereas the diameter including the lipid belt is ∼12 nm.We resolved 24 lipid molecules in the central cavity patch, andan overall total of 31 complete lipid molecules plus an additional11 individual alkyl chains that could be from other lipid mole-cules. We found no evidence of ordered SMA molecules.

The patch of lipid bilayer in the central cavity is shaped like atriangular two-layer cake (Fig. 2 A–D) with each side facing theTM domain of a particular subunit (Fig. 2E). The overall shapesand orientations of the leaflet on the periplasmic side (outerleaflet) and of the cytosol-facing leaflet (inner leaflet) are dis-tinctly different (Fig. 2 B–D). Twelve lipid molecules, built asthe predominant bacterial lipid phosphatidylethanolamine (PE)(33), could be fitted into the EM density of each leaflet (Fig. 2 Cand D). The thickness of the lipid bilayer is ∼31 Å, as calculatedfrom the averaged Z coordinates for phosphorus atoms of themodeled lipid molecules.

Fig. 1. EM density of AcrB in a native cell-membrane nanoparticle. (A andB) Surfaces of EM density features. Gray-colored surfaces show density fea-tures covered by the protein model for the AcrB trimer. Yellow-coloredsurfaces show remaining density features, presumed to be from lipids. Thedensity within the central cavity between AcrB TM domains is interpreted asa patch of lipid bilayer. (A) Side view, as seen from within the membrane. (B)Bottom view, as seen from the cytoplasm. (C) Ribbon diagram of the AcrBtrimer (L-state chain A: cyan; T-state chain B: orange; O-state chain C: gray)with superimposed EM density in the central cavity (yellow). (D) Enlargedview of boxed region of EM density for the native cell-membrane lipidbilayer.

Fig. 2. Features of the lipid bilayer from the central cavity. (A) Side view ofthe EM density of lipid-bilayer structure (blue). The patch of the lipid bilayerlooks like a triangular two-layer cake. Lipid molecules fitted at the apices ofeach triangle are drawn in red. (B) Lipid bilayer as viewed from the peri-plasmic space (top view). The outer leaflet (orange) is rotated ∼10° clockwiserelative to the inner leaflet (blue). (C and D) EM density with superimposedlipid models for the outer leaflet (C) and inner leaflet (D). Here, both EMmaps are colored blue, but distinctive triangular shapes relate C to orangeand D to blue in B. The lipid model is in stick representation with coloredphosphorus (orange), carbon (gray), and nitrogen (blue). (E) Stereoview ofthe central cavity region of AcrB viewed as in B. Protein ribbons are coloredas in Fig. 1C. The phosphoryl heads from lipids 1, 5, and 9 are at the cyto-plasmic surface of the inner leaflet of the bilayer, and those from lipids 13,17, and 21 are at the periplasmic surface of the outer leaflet. Triangles thatconnect phosphorous atoms in these two sets of apical lipids are drawn toemphasize differences in size, shape, and orientation for the two leaflets.

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A striking feature of the lipid bilayer is that the outer triangle oflipids is rotated relative to the inner triangle by ∼10° clockwise, asviewed from the periplasm (Fig. 2 B–D). Whereas the inner leaflethas lipid molecules packed quite tightly, there are some spacesbetween lipids within the outer leaflet, but these are occupied byprotein side chains (Lipids Interact Extensively with AcrB Protein,both in the Central Cavity and in the Belt Outside). Thus, in terms oflipids alone, the outer leaflet is larger than the inner one (Fig. 2 Cand D). A particular lipid molecule is at each triangular apex, andthe distances between the phosphorus atoms at these apices are,respectively, 26.8, 25.4, and 22.4 Å for the inner leaflet and 28.5,29.9, and 33.4 Å for the outer leaflet (Fig. 2E). Thereby, thesetriangles have areas of 263.5 and 399.7 Å2, respectively. Thephospholipids of the inner leaflet are disposed in a regular pat-tern with alkyl tails mostly straight; outer-leaflet phospholipidsare also ordered, meaning that they are defined by distinct den-sity features, but their alkyl tails are mostly curved (Figs. 2 C andD and 3 A–C).

Lipids Interact Extensively with AcrB Protein, both in the CentralCavity and in the Belt Outside. We found several specific hydro-phobic protein–lipid interactions in the central cavity. A385,F386, and F458 from each protomer all protrude into the outerleaflet (Fig. 3B), occupying spaces left from disrupted packing ofthe lipid tails (Fig. 2C). As well, M1, F4, F11, and M447 fromeach protomer interact with the inner leaflet (Fig. 3D and SIAppendix, Fig. S3). In addition, certain lipid head groups interactwith the protein through hydrogen bonding. In particular, theguanidyl group of R8 on subunit B (T state) hydrogen bonds(3.1 Å) with the phosphoryl group of lipid 9 in the inner leaflet(Fig. 3E and SI Appendix, Fig. S4E), and the corresponding R8on subunit C (O state) is at possible hydrogen-bonding distance(3.4 Å) to the phosphoryl group of lipid 5 (SI Appendix, Fig.S4F); however, R8 of subunit A (L state) is too far (closest

distance of 4.3 Å) from the phosphoryl group of correspondinglipid 1 (SI Appendix, Fig. S4D). In the outer leaflet, the phos-phoryl group from lipid 13 hydrogen bonds (3.1 Å) with thebackbone N of G460 from subunit C (SI Appendix, Fig. S4I);however, corresponding lipids 17 and 21 are too distant fromtheir corresponding F459–G460–G461 segments for hydrogenbonding (SI Appendix, Fig. S4 G and H). The distinctions in lipidinteractions with the AcrB trimer reflect the intrinsic asymmetryof AcrB and its associated lipid bilayer.The lipid belt surrounding the TM region of AcrB is generally

much less ordered than the lipid-filled central cavity, but somelipid molecules do interact directly with outward-facing TM he-lices. For example, the phosphoryl group of lipid A hydrogenbonds (3.3 Å) to Nδ of H338 from subunit A (Fig. 3F and SIAppendix, Fig. S5A). Lipid B is similarly disposed in relation toH338 from subunit B; however, this interaction seems slightly toolong for hydrogen bonding (SI Appendix, Fig. S4B). Lipid C oc-cupies an analogous site in relation to subunit C, but here H338is remote from the head group (SI Appendix, Fig. S4C). Besidesthose lipid molecules nearby to H338, several other lipid-beltlipid molecules also make hydrophobic interactions with outerhelices from TM domains (SI Appendix, Fig. S5).Intimate lipid–protein interactions were also observed in 2D

crystals of aquaporin-0 (34), although in this case the system wasreconstituted from 1,2-Dimyristoyl-sn-glycero-3-phosphocholinelipids, whereas our AcrB particles were extracted directly fromnatural membranes.

Lipid-Bilayer Function in Harmonizing Peristaltic ConformationalChanges Through AcrB Trimers. Whereas lipid tails in the outerlayer are irregularly curved and loosely packed, those in the innerlayer are relatively straight and quite close-packed (Fig. 3A). Incross-section, midway through the inner leaflet, the EM densityshows a hexagonal pattern (Fig. 4A) that is remarkably similar to

Fig. 3. Structure of lipid bilayer and protein–lipid interactions. (A) Atomic model of the lipid bilayer in stick representation with coloring of phosphorus(orange), oxygen (red), nitrogen (blue), and carbon (gray). (B) Atomic model of the outer leaflet showing 12 lipid molecules (sticks) and protein residues thatprotrude from each subunit into the lipid array. Protein residues are colored as in Fig. 1C: subunits A (cyan), B (orange), and C (gray). (C) Atomic model of 12lipid molecules in the inner leaflet. (D) Hydrophobic interactions in the central cavity between the lipid bilayer (yellow density surfaces) and AcrB subunit A.A385, F386, and F458 interact with the outer leaflet, and M1, F4, F11, and M447 do so with the inner leaflet. Subunits B and C make similar but distinctinteractions at other faces of the triangular bilayer patch. (E) Interactions of residue R8 from AcrB subunit B (T state) with lipid 9 from the inner leaflet.Hydrogen bonding between the guanidyl and phosphoryl groups is indicated. (F) Interactions of residue H338 with lipid A from the lipid belt surrounding TMdomains of the AcrB trimer.

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the pattern in a PE crystal structure (2), predicted 40 y ago to reflectnatural membranes; however, the real lipid cell-membrane struc-ture is much more complex than that of artificial lipid structure. Wenumbered the 12 PE lipid molecules in the inner leaflet, 1–12, andwe identify associated alkyl chains in the grid with these numbers(Fig. 4B). Protein also contributes to the hexagonal pattern; forexample, densities for M1 from each of the subunits and F4 fromsubunit A (L state) are seen in Fig. 4C, and M1 is actually cutthrough in the cross-section. For 10 of the 12 PE molecules, thehead-group density is well defined and the two pair of unconnectedlipid tails are adjacent; thus, all 24 lipid tails are assigned to headgroups of specific lipid molecules as drawn in Fig. 4D.Natural lipid bilayers are fluid and they can adapt, albeit with

certain resistance, to conformational changes in associated pro-teins. AcrB itself takes on multiple states. The three subunits inasymmetric AcrB structures (28, 29, 32) are distinct, both fromone another and also from the conformation in symmetric AcrB(25). This distinction has implications for the lipid bilayer that weexpect will occupy the central cavity for all of these states whenin a natural membrane. The lipid structure must accommodateall of these protein states. In Fig. 4E, we present a lipid arrayreorganized into threefold symmetry from the one observed inour EM structure, and we include paths of transformation thatcan move from the lipid array back into the asymmetric array ofFig. 4D. The proposed transport mechanism for AcrB (32) haseach protomer moving successively and in coordination fromstate L through T into O and then back to L (L → T → O → L).As these protein movements occur, the lipid structure must alsomove to accommodate.

The net result for the working system gives the appearance of120° rotations at each step; in fact, however, subunits stay inplace while undergoing the succession of conformationalchanges, which in turn must be accompanied by shifts in the lipidstructure, as envisioned in SI Appendix, Fig. S7. From examinationof the extensive protein–lipid interactions in AcrB (Fig. 3D and SIAppendix, Figs. S3 and S4), we propose that the lipid bilayer in thecentral cavity serves to harmonize conformational changes in theperistaltic mechanism of drug extrusion by AcrB. Through definedprotein contacts, the lipid bilayer senses the conformationalchanges that occur in each TM domain and then transduceseffects of these changes through the lipid bilayer to neigh-boring protomers in a viscous interplay between cavity lipidsand the AcrB trimer. This process happens reciprocally, suchas to synchronize movement of client drugs through the pseu-dorotatory AcrB trimer.

Lipid Bilayer in the AcrB D407A Trimer Mutant. The TM domain ofAcrB contains conserved amino acid residues proposed to beimportant for a proton relay mechanism in the drug/protonantiport activity. Crystal structures of AcrB with mutated proton-relay residues, D407A, D408A, K940A, and T978A, all showed adramatic collapse of TM domains toward the central cavity (35).Most noticeably, the distances between F386 positions droppedfrom 17.6 Å in wild-type AcrB to 6.7 Å for mutant AcrB D407Afor the detergent-solubilized protein in crystal structures (Fig.5E). Similar TM shifts occurred for the other mutant proteins.To test importance of the lipid bilayer for the structure and

activity of AcrB, in light of the remarkably large changesreported previously, we also determined the cryo-EM structure

Fig. 4. Hexagonal pattern within the lipid bilayer. (A and B) EM density drawn as a solid yellow surface and sliced through the inner leaflet. Sliced surfaces inA are colored red. Lines of a hexagonal grid are drawn in yellow. Hydrocarbon tails in B are numbered by lipid identifiers 1–12. Density for protein residue M1also occupies a grid position in this cut. (C) Stereoview of the EM density of the inner leaflet of the lipid bilayer. (D) Hexagonal grid as in A. The red dotrepresents the phosphoryl head, green is for a tail on glycerol position C2, purple is for a tail on glycerol position C3, and yellow is for unassigned positions. (E)Proposed hexagonal pattern for the inner leaflet in a C3-symmetric AcrB trimer. The red triangle marks the threefold axis position. Blue arrow-directed linesspecify translations and rotations in lipid positions that can shift this symmetric pattern into the asymmetric pattern as that of D. (F) Correspondence of lipidnumberings in the asymmetric lipid array (D) with those in the symmetric lipid array (E).

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for SMA-extracted AcrB D407A trimer at 3.0-Å resolution. Asfor wild-type AcrB, the AcrB D407A structure has a similar lipid-bilayer patch located in its central cavity. As for wild-type AcrB,lipid hydrocarbon tails in this inner leaflet are also hexagonallyarranged (Fig. 5A), and we observed similar hydrophobic inter-actions between AcrB and the lipid bilayer (Fig. 5B). In com-parison with the structure of the wild-type AcrB trimer, theamino acid residues that interact with the lipid bilayer are rela-tively unmoved (Figs. 3D and 5B). Consistent with this obser-vation, the r.m.s.d. value is less than 1 Å in a comparisonbetween the wild-type AcrB and AcrB D407 using all Cαatoms.The role of the conserved residues, and of the lipid bilayer, in

the proton relay mechanism for AcrB needs further study. We doobserve changes in the D407A mutant structure, and also in itslipid bilayer, compared with our wild-type AcrB structure (Fig. 5C and D); however, these changes are subtle compared with thedramatic shifts seen for proton relay mutants in the detergent-extracted situation (Fig. 5 E and F). The likely factor leading tocollapse of TM domains in crystal structures of the mutants is theabsence of the supporting lipid bilayer because of the use ofdetergents. The lipid bilayer, as preserved in the central cavityafter SMA extraction, provides a restraining structural supportfor the TM domains (Fig. 5B). The tight packing of lipid mole-cules in the inner leaflet also suggests that the central cavity isnot part of the drug-transport pathway.

Prospects for Native Cell Membrane Nanoparticles. Detergents havebeen essential for advances in membrane-protein structural bi-ology, but they also have limitations because the lipid bila-yers that detergent solubilization destroys may be crucial formembrane-protein function and stability. The best niche for amembrane protein is in its native cell-membrane environment.The system that we have been developing for detergent-freemembrane-protein solubilization into native cell-membranenanoparticles appears to preserve much of the natural lipid

environment. From this cryo-EM analysis of AcrB as solubilizeddirectly with SMA copolymer, we could build a total of 31 lipidmolecules and 11 additional hydrocarbon chains that likely de-rive from lipids. Most remarkably, the central cavity betweenAcrB TM domains sustains a 24-lipid patch of mostly well-ordered bilayer structure. Regularity in the hexagonal pattern ofthe inner leaflet is similar to that in a PE crystal structure (2),and this regularity contrasts with highly irregular packing in theouter leaflet. Protein side chains interact with both leaflets andparticipate in the hexagonal pattern. Lipid ordering in the proteinconfines of the AcrB central cavity may be a special situation, butwe also see well-ordered lipid structure in the surrounding lipidbelt, even with our relatively unsophisticated analysis of the map.A system such as ours for preparing native cell-membrane

nanoparticles has certain advantages. First, and most impor-tantly, a protein can be extracted with its native local membranestructure largely intact. Whereas apolipodisc (36), bicelle (37), orsaposin (38) alternatives need to include detergents at somestage, here we could remain truly detergent-free. Second, thenative cell-membrane nanoparticle system might catch membrane-protein complexes that are labile in detergents, as was demon-strated for a plant metabolon (39). Lastly, this detergent-freesystem is well suited for single-particle cryo-EM analysis, pro-viding evenly distributed particles. Our current native cell-membranenanoparticle system still has shortcomings. Not all tested membraneproteins performed well. However, the system has much scope forimprovement, and we expect a very positive impact on membrane-protein research.

Materials and MethodsPolymers preparation, protein expression, purification, and structure determi-nation protocols are described in SI Appendix, SI Materials and Methods.

ACKNOWLEDGMENTS. The Y.G. laboratory is supported by the VirginiaCommonwealth University (VCU) School of Pharmacy and Department ofMedicinal Chemistry, through startup funds, and by the VCU Institute forStructural Biology, Drug Discovery and Development, through laboratory

Fig. 5. Lipid bilayer in AcrB D407A trimer prevents large distance movement of F386. (A) EM density drawn as solid yellow surface and sliced through theinner leaflet (red cross-sections). The hydrocarbon tails show a hexagonal pattern as in Fig. 4A. (B) Hydrophobic interactions in the central cavity between thelipid bilayer (yellow density surface) and AcrB subunit A. A385, F386, and F458 interact with the outer leaflet, and M1, F4, and M447 interact with the innerleaflet. Subunits B and C interact similarly but distinctly at the other faces of the triangular bilayer patch. (C) EM density and conformation of A407, D408,K940, N941, and T978 within AcrB D407A mutant. K940 and T978 form a hydrogen bond with a distance of 3.3 Å; K940 and N941 form a hydrogen bond witha distance of 2.5 Å. (D) EM density and conformation of D407A, D408, K940, N-941, and T978 within wild-type AcrB. K940 forms a hydrogen bond with D407with a distance of 3.5 Å; K940 also forms a hydrogen bond with T978 with a distance of 3.4 Å. (E) Stick model of AcrB D407A for F386 residues superimposedon space-filing wild-type AcrB residues. (F) F386 residues from the crystal structure of detergent-extracted D407A AcrB (magenta) compared with those fromthe wild-type crystal structure (cyan). F386–F386 distances shift from 17.6 Å down to 6.7 Å as shown. For reference, the stick model for F386 residues in thecryo-EM model of wild-type AcrB (white) is also copied here from E.

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space and facilities. This research was also supported, in part, by the HowardHughes Medical Institute and NIH Grant R01 GM29169 (to J.F.), by PublicHealth Service Grants DA024022 and DA044855 (to Y.Z.), and by NIH GrantsR01 GM107462 and P41 GM116799 (to W.A.H.). We thank Klaas Martinus Posfor the AcrB expression plasmid; Bill Rice and Ed Eng for help in data collectionon Titan Krios #2 at the Simons ElectronMicroscopy Center, which is supported

by NIH Grants GM103310 and S10 OD019994-01 and the Simons Foundation(349247) to Bridget Carragher and Clint Potter; and Ravi C. Kalathur, RenatoBruni, Brian Kloss, and Filippo Mancia for constructive comments and adviceon nanoparticle systems from the Center on Membrane Protein Productionand Analysis, which is supported at the New York Structural Biology Center byNIH Grant P41 GM116799.

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