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Home , Lipid bilayer, Membrane lipid, Membrane transport

Structure and activity of bilayer within a - transporter

Weihua 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 , Drug Discovery and Development, Virginia Commonwealth University, Richmond, VA 23219; cIntegrated Program in Cellular, Molecular, and Biomedical Studies, Columbia University, New York, NY 10032; dDepartment of and Molecular Biophysics, Columbia University, New York, NY 10032; eDepartment of Biological 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 function in native , but extrac- 1.9 Å (30). Nevertheless, the mechanism of is still tion into isolated particles is needed for many biochemical and far from clear, in part because crucial structural information re- structural analyses. Commonly used detergent-extraction meth- garding protein–lipid interaction is missing (31). The AcrB trimer ods destroy naturally associated lipid bilayers. Here, we devised a has a central cavity between transmembrane (TM) domains of the detergent-free method for preparing cell-membrane nanopar- three protomers, where a portion of may exist (26). ticles to study the multidrug exporter AcrB, by cryo-EM at 3.2-Å Although detergent and some chains have been resolution. We discovered a remarkably well-organized lipid- identified, organized lipid structure has eluded detection in the bilayer structure associated with transmembrane domains of the central cavity or elsewhere. AcrB trimer. This bilayer patch comprises 24 lipid molecules; inner We have developed a native cell-membrane nanoparticles leaflet chains are packed in a hexagonal array, whereas the outer system based on the previously reported SMALP method (18) leaflet has highly irregular but ordered packing. Protein side chains for high-resolution structure determination using single-particle interact with both leaflets and participate in the hexagonal pattern. cryo-EM. Here, we report our discovery of a high-resolution We suggest that the lipid bilayer supports and harmonizes peristal- structure of lipid bilayer in extracted nanoparticles of AcrB. tic motions through AcrB trimers. In AcrB D407A, a putative proton- relay mutant, lipid bilayer buttresses protein interactions lost in The structure of the lipid bilayer and its interaction with AcrB crystal structures after detergent-solubilization. Our detergent-free provide us with important insights both for understanding the system preserves lipid–protein interactions for visualization and should be broadly applicable. Significance

AcrB | cryo-EM | nanoparticle | | styrene maleic Membrane proteins function naturally as imbedded in the lipid copolymer bilayers of cell membranes, but isolation into homogeneous and soluble preparations is needed for many biochemical ell membranes and their constituent proteins are crucial for studies. Detergents, which are used traditionally to extract and Cliving , and great efforts have been made to un- purify membrane proteins from cells, also remove most derstand the structures of cell-membrane systems (1–8). De- protein-associated lipid molecules as they disrupt the mem- tergent solubilization has dominated membrane-protein studies branes. We have devised a detergent-free system to prepare (9, 10); however, detergents have significant drawbacks because native cell-membrane nanoparticles for biochemical analysis. In they destroy cell membranes and remove protein-associated lipid application to the membrane transporter AcrB, we demon- molecules (11, 12). Protein–lipid interactions play crucial roles strate that these detergent-free nanoparticles are suitable for for membrane proteins; for example, activity of mitochondrial cryo-EM imaging at high resolution and that the natural lipid- respiratory complex I extracted with detergents suffers exten- bilayer structure so preserved is important for the functional sively from the depletion of lipid components (13, 14). The integrity of AcrB. This nanoparticle system should be broadly importance of the protein–lipid interactions in biology and applicable in membrane-protein research. medicine fosters the need for procedures that preserve while extracting proteins from membranes. 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; Membrane-active polymers such as styrene maleic acid (SMA) R.A.G. set up and maintained EM facilities;Y.Z.gaveadviceonchemicalsynthesis; copolymer, diisobutylene maleic acid copolymer, and others have W.Q., G.G.X., Y.Z., and Y.G. contributed new reagents/analytic tools; W.Q., Z.F., W.A.H., been shown to be useful in membrane studies (15–17). Extrac- and Y.G. analyzed data; W.A.H. gave advice on structure analysis; W.Q., Z.F., G.G.X., tion of membrane proteins into SMA particles R.A.G., Y.Z., J.F., W.A.H., and Y.G. wrote the paper. (SMALPs) was demonstrated first from proteoliposomes (16), Reviewers: Y.C., University of California, San Francisco; and M.C.W., University of Virginia. but similar procedures also permit direct solubilization from The authors declare no conflict of interest. cell membranes, never employing detergents (18). SMA co- Published under the PNAS license. polymer has emerged as an alternative to traditional detergents Data deposition: Three-dimensional density maps and atomic models have been depos- for membrane-protein research (18–21), including use in struc- ited in the Microscopy Data Bank, www.ebi.ac.uk/pdbe/emdb [EMDB entry nos. tural analysis (22, 23). A recent cryo-EM analysis of SMA- 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 and extracted AcrB reached a resolution limit of 8.8 Å (24). lipid bilayer)], and the Protein Data Bank, www.wwpdb.org [PDB ID codes 6BAJ (wild- AcrB is an archetypal resistance-nodulation-division multidrug type AcrB and lipid bilayer) and 6CSX (AcrB D407A mutant and lipid bilayer)]. exporter from the inner of gram-negative bacte- 1W.Q. and Z.F. contributed equally to this work. Escherichia coli ria. Crystal structures of AcrB from were first 2To whom correspondence may be addressed. Email: [email protected], wah2@ reported as symmetric trimers (25–27) and later as asymmetric cumc.columbia.edu, or [email protected]. trimers (28, 29). Because of its biological and biomedical im- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

portance, AcrB has been investigated extensively and many AcrB 1073/pnas.1812526115/-/DCSupplemental. BIOPHYSICS AND

structures have been reported, having resolutions as high as Published online December 3, 2018. COMPUTATIONAL BIOLOGY

www.pnas.org/cgi/doi/10.1073/pnas.1812526115 PNAS | December 18, 2018 | vol. 115 | no. 51 | 12985–12990 Downloaded by guest on September 29, 2021 active mechanism of this transporter and for understanding protein–lipid interactions in cell membranes generally. Results and Discussion Lipid Bilayer Ordering in Native Cell-Membrane Particles of AcrB. We prepared native cell-membrane nanoparticles of E. coli AcrB using membrane-active SMA polymers. The nanoparticles were purified by single-step Ni-affinity chromatography, applied directly to grids, and vitrified for single-particle cryo-EM analysis. A 3D reconstruction with C1 symmetry achieved a final density map of 3.2-Å resolution (Fig. 1 A and B and SI Appendix,Fig.S1). We initially tried to reconstruct the 3D EM map in C3 symmetry; however, that density map was fragmented, especially so in the TM region, and we could not see lipid-bilayer structure in the central cavity. The C1 reconstruction was fitted by an asymmetric AcrB trimer (Fig. 1C and SI Appendix,Fig.S2), where each pro- tomer exists in a distinct state (L for loose, binding-ready; T for tight, substrate-bound; and O for open, substrate release) as in the asymmetric crystal structures (28, 29, 32), but here in this cryo-EM structure with differences from corresponding subunits in the crystal 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 diameter of ∼9 nm, whereas the diameter including the lipid belt is ∼12 nm. We resolved 24 lipid molecules in the central cavity patch, and an overall total of 31 complete lipid molecules plus an additional 11 individual alkyl chains that could be from other lipid - cules. We found no evidence of ordered SMA molecules.

Fig. 2. Features of the lipid bilayer from the central cavity. (A) Side view of the EM density of lipid-bilayer structure (blue). The patch of the lipid bilayer looks like a triangular two-layer cake. Lipid molecules fitted at the apices of each triangle are drawn in red. (B) Lipid bilayer as viewed from the peri- plasmic space (top view). The outer leaflet (orange) is rotated ∼10° clockwise relative to the inner leaflet (blue). (C and D) EM density with superimposed lipid models for the outer leaflet (C) and inner leaflet (D). Here, both EM maps are colored blue, but distinctive triangular shapes relate C to orange and D to blue in B. The lipid model is in stick representation with colored phosphorus (orange), (gray), and nitrogen (blue). (E) Stereoview of the central cavity region of AcrB viewed as in B. Protein ribbons are colored as 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 that connect phosphorous atoms in these two sets of apical lipids are drawn to emphasize differences in size, shape, and orientation for the two leaflets.

The patch of lipid bilayer in the central cavity is shaped like a triangular two-layer cake (Fig. 2 A–D) with each side facing the Fig. 1. EM density of AcrB in a native cell-membrane nanoparticle. (A and E B) Surfaces of EM density features. Gray-colored surfaces show density fea- TM domain of a particular subunit (Fig. 2 ). The overall shapes tures covered by the protein model for the AcrB trimer. Yellow-colored and orientations of the leaflet on the periplasmic side (outer surfaces show remaining density features, presumed to be from lipids. The leaflet) and of the -facing leaflet (inner leaflet) are dis- density within the central cavity between AcrB TM domains is interpreted as tinctly different (Fig. 2 B–D). Twelve lipid molecules, built as a patch of lipid bilayer. (A) Side view, as seen from within the membrane. (B) the predominant bacterial lipid (PE) Bottom view, as seen from the . (C) Ribbon diagram of the AcrB (33), could be fitted into the EM density of each leaflet (Fig. 2 C trimer (L-state chain A: cyan; T-state chain B: orange; O-state chain C: gray) D ∼ with superimposed EM density in the central cavity (yellow). (D) Enlarged and ). The thickness of the lipid bilayer is 31 Å, as calculated view of boxed region of EM density for the native cell- from the averaged Z coordinates for phosphorus atoms of the bilayer. modeled lipid molecules.

12986 | www.pnas.org/cgi/doi/10.1073/pnas.1812526115 Qiu et al. Downloaded by guest on September 29, 2021 A striking feature of the lipid bilayer is that the outer triangle of distance of 4.3 Å) from the phosphoryl group of corresponding lipids is rotated relative to the inner triangle by ∼10° clockwise, as lipid 1 (SI Appendix, Fig. S4D). In the outer leaflet, the phos- viewed from the periplasm (Fig. 2 B–D). Whereas the inner leaflet phoryl group from lipid 13 hydrogen bonds (3.1 Å) with the has lipid molecules packed quite tightly, there are some spaces backbone N of G460 from subunit C (SI Appendix, Fig. S4I); between lipids within the outer leaflet, but these are occupied by however, corresponding lipids 17 and 21 are too distant from protein side chains (Lipids Interact Extensively with AcrB Protein, their corresponding F459–G460–G461 segments for hydrogen both in the Central Cavity and in the Belt Outside). Thus, in terms of bonding (SI Appendix, Fig. S4 G and H). The distinctions in lipid lipids alone, the outer leaflet is larger than the inner one (Fig. 2 C interactions with the AcrB trimer reflect the intrinsic asymmetry and D). A particular lipid is at each triangular apex, and of AcrB and its associated lipid bilayer. the distances between the phosphorus atoms at these apices are, The lipid belt surrounding the TM region of AcrB is generally respectively, 26.8, 25.4, and 22.4 Å for the inner leaflet and 28.5, much less ordered than the lipid-filled central cavity, but some 29.9, and 33.4 Å for the outer leaflet (Fig. 2E). Thereby, these lipid molecules do interact directly with outward-facing TM he- triangles have areas of 263.5 and 399.7 Å2,respectively.The lices. For example, the phosphoryl group of lipid A hydrogen of the inner leaflet are disposed in a regular pat- bonds (3.3 Å) to Nδ of H338 from subunit A (Fig. 3F and SI tern with alkyl tails mostly straight; outer-leaflet phospholipids Appendix, Fig. S5A). Lipid B is similarly disposed in relation to are also ordered, meaning that they are defined by distinct den- H338 from subunit B; however, this interaction seems slightly too sity features, but their alkyl tails are mostly curved (Figs. 2 C and long for hydrogen bonding (SI Appendix, Fig. S4B). Lipid C oc- D and 3 A–C). cupies an analogous site in relation to subunit C, but here H338 is remote from the head group (SI Appendix, Fig. S4C). Besides Lipids Interact Extensively with AcrB Protein, both in the Central those lipid molecules nearby to H338, several other lipid-belt Cavity and in the Belt Outside. We found several specific hydro- lipid molecules also make hydrophobic interactions with outer phobic protein–lipid interactions in the central cavity. A385, helices from TM domains (SI Appendix, Fig. S5). F386, and F458 from each protomer all protrude into the outer Intimate lipid–protein interactions were also observed in 2D leaflet (Fig. 3B), occupying spaces left from disrupted packing of crystals of -0 (34), although in this case the system was the lipid tails (Fig. 2C). As well, M1, F4, F11, and M447 from reconstituted from 1,2-Dimyristoyl-sn-glycero-3-phosphocholine each protomer interact with the inner leaflet (Fig. 3D and SI lipids, whereas our AcrB particles were extracted directly from Appendix, Fig. S3). In addition, certain lipid head groups interact natural membranes. with the protein through hydrogen bonding. In particular, the guanidyl group of R8 on subunit B (T state) hydrogen bonds Lipid-Bilayer Function in Harmonizing Peristaltic Conformational (3.1 Å) with the phosphoryl group of lipid 9 in the inner leaflet Changes Through AcrB Trimers. Whereas lipid tails in the outer (Fig. 3E and SI Appendix, Fig. S4E), and the corresponding R8 layer are irregularly curved and loosely packed, those in the inner on subunit C (O state) is at possible hydrogen-bonding distance layer are relatively straight and quite close-packed (Fig. 3A). In (3.4 Å) to the phosphoryl group of lipid 5 (SI Appendix, Fig. cross-section, midway through the inner leaflet, the EM density S4F); however, R8 of subunit A (L state) is too far (closest shows 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 that protrude 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 12 lipid 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 distinct interactions 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 TM BIOPHYSICS AND

domains of the AcrB trimer. COMPUTATIONAL BIOLOGY

Qiu et al. PNAS | December 18, 2018 | vol. 115 | no. 51 | 12987 Downloaded by guest on September 29, 2021 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 in A are colored red. Lines of a hexagonal grid are drawn in yellow. tails in B are numbered by lipid identifiers 1–12. Density for protein residue M1 also 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 dot represents 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 lines specify translations and rotations in lipid positions that can shift this symmetric pattern into the asymmetric pattern as that of D.(F) Correspondence of lipid numberings in the asymmetric lipid array (D) with those in the symmetric lipid array (E).

the pattern in a PE crystal structure (2), predicted 40 y ago to reflect The net result for the working system gives the appearance of natural membranes; however, the real lipid cell-membrane struc- 120° rotations at each step; in fact, however, subunits stay in ture is much more complex than that of artificial lipid structure. We place while undergoing the succession of conformational numbered the 12 PE lipid molecules in the inner leaflet, 1–12, and changes, which in turn must be accompanied by shifts in the lipid we identify associated alkyl chains in the grid with these numbers structure, as envisioned in SI Appendix,Fig.S7. From examination (Fig. 4B). Protein also contributes to the hexagonal pattern; for of the extensive protein–lipid interactions in AcrB (Fig. 3D and SI example, densities for M1 from each of the subunits and F4 from Appendix, Figs. S3 and S4), we propose that the lipid bilayer in the subunit A (L state) are seen in Fig. 4C, and M1 is actually cut central cavity serves to harmonize conformational changes in the through in the cross-section. For 10 of the 12 PE molecules, the peristaltic mechanism of drug extrusion by AcrB. Through defined head-group density is well defined and the two pair of unconnected protein contacts, the lipid bilayer senses the conformational lipid tails are adjacent; thus, all 24 lipid tails are assigned to head changes that occur in each TM domain and then transduces groups of specific lipid molecules as drawn in Fig. 4D. effects of these changes through the lipid bilayer to neigh- Natural lipid bilayers are fluid and they can adapt, albeit with boring protomers in a viscous interplay between cavity lipids certain resistance, to conformational changes in associated pro- and the AcrB trimer. This process happens reciprocally, such teins. AcrB itself takes on multiple states. The three subunits in as to synchronize movement of client drugs through the pseu- asymmetric AcrB structures (28, 29, 32) are distinct, both from dorotatory AcrB trimer. one another and also from the conformation in symmetric AcrB Lipid Bilayer in the AcrB D407A Trimer Mutant. The TM domain of (25). This distinction has implications for the lipid bilayer that we AcrB contains conserved residues proposed to be expect will occupy the central cavity for all of these states when important for a proton relay mechanism in the drug/proton in a natural membrane. The lipid structure must accommodate antiport activity. Crystal structures of AcrB with mutated proton- E all of these protein states. In Fig. 4 , we present a lipid array relay residues, D407A, D408A, K940A, and T978A, all showed a reorganized into threefold symmetry from the one observed in dramatic collapse of TM domains toward the central cavity (35). our EM structure, and we include paths of transformation that Most noticeably, the distances between F386 positions dropped can move from the lipid array back into the asymmetric array of from 17.6 Å in wild-type AcrB to 6.7 Å for mutant AcrB D407A Fig. 4D. The proposed transport mechanism for AcrB (32) has for the detergent-solubilized protein in crystal structures (Fig. each protomer moving successively and in coordination from 5E). Similar TM shifts occurred for the other mutant proteins. state L through T into O and then back to L (L → T → O → L). To test importance of the lipid bilayer for the structure and As these protein movements occur, the lipid structure must also activity of AcrB, in light of the remarkably large changes move to accommodate. reported previously, we also determined the cryo-EM structure

12988 | www.pnas.org/cgi/doi/10.1073/pnas.1812526115 Qiu et al. Downloaded by guest on September 29, 2021 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 the inner leaflet (red cross-sections). The hydrocarbon tails show a hexagonal pattern as in Fig. 4A.(B) Hydrophobic interactions in the central cavity between the lipid 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 inner leaflet. 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 with a distance of 3.3 Å; K940 and N941 form a hydrogen bond with a 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 D407 with 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 superimposed on space-filing wild-type AcrB residues. (F) F386 residues from the crystal structure of detergent-extracted D407A AcrB (magenta) compared with those from the 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 the cryo-EM model of wild-type AcrB (white) is also copied here from E.

for SMA-extracted AcrB D407A trimer at 3.0-Å resolution. As environment. From this cryo-EM analysis of AcrB as solubilized for wild-type AcrB, the AcrB D407A structure has a similar lipid- directly with SMA copolymer, we could build a total of 31 lipid bilayer patch located in its central cavity. As for wild-type AcrB, molecules and 11 additional hydrocarbon chains that likely de- lipid hydrocarbon tails in this inner leaflet are also hexagonally rive from lipids. Most remarkably, the central cavity between arranged (Fig. 5A), and we observed similar hydrophobic inter- AcrB TM domains sustains a 24-lipid patch of mostly well- actions between AcrB and the lipid bilayer (Fig. 5B). In com- ordered bilayer structure. Regularity in the hexagonal pattern of parison with the structure of the wild-type AcrB trimer, the the inner leaflet is similar to that in a PE crystal structure (2), amino acid residues that interact with the lipid bilayer are rela- and this regularity contrasts with highly irregular packing in the tively unmoved (Figs. 3D and 5B). Consistent with this obser- outer leaflet. Protein side chains interact with both leaflets and vation, the r.m.s.d. value is less than 1 Å in a comparison participate in the hexagonal pattern. Lipid ordering in the protein between the wild-type AcrB and AcrB D407 using all Cα confines of the AcrB central cavity may be a special situation, but atoms. we also see well-ordered lipid structure in the surrounding lipid The role of the conserved residues, and of the lipid bilayer, in belt, even with our relatively unsophisticated analysis of the map. the proton relay mechanism for AcrB needs further study. We do A system such as ours for preparing native cell-membrane observe changes in the D407A mutant structure, and also in its nanoparticles has certain advantages. First, and most impor- lipid bilayer, compared with our wild-type AcrB structure (Fig. 5 tantly, a protein can be extracted with its native local membrane C and D); however, these changes are subtle compared with the structure largely intact. Whereas apolipodisc (36), bicelle (37), or dramatic shifts seen for proton relay mutants in the detergent- saposin (38) alternatives need to include detergents at some extracted situation (Fig. 5 E and F). The likely factor leading to stage, here we could remain truly detergent-free. Second, the collapse of TM domains in crystal structures of the mutants is the native cell-membrane nanoparticle system might catch membrane- absence of the supporting lipid bilayer because of the use of protein complexes that are labile in detergents, as was demon- detergents. The lipid bilayer, as preserved in the central cavity strated for a plant metabolon (39). Lastly, this detergent-free after SMA extraction, provides a restraining structural support system is well suited for single-particle cryo-EM analysis, pro- for the TM domains (Fig. 5B). The tight packing of lipid mole- viding evenly distributed particles. Our current native cell-membrane cules in the inner leaflet also suggests that the central cavity is nanoparticle system still has shortcomings. Not all tested membrane not part of the drug-transport pathway. proteins performed well. However, the system has much scope for improvement, and we expect a very positive impact on membrane- Prospects for Native Cell Membrane Nanoparticles. Detergents have protein research. been essential for advances in membrane-protein structural bi- ology, but they also have limitations because the lipid bila- Materials and Methods yers that detergent solubilization destroys may be crucial for Polymers preparation, protein expression, purification, and structure determi- membrane-protein function and stability. The best niche for a nation protocols are described in SI Appendix, SI Materials and Methods. is in its native cell-membrane environment. The system that we have been developing for detergent-free ACKNOWLEDGMENTS. The Y.G. laboratory is supported by the Virginia Commonwealth University (VCU) School of Pharmacy and Department of membrane-protein solubilization into native cell-membrane Medicinal Chemistry, through startup funds, and by the VCU Institute for BIOPHYSICS AND

nanoparticles appears to preserve much of the natural lipid Structural Biology, Drug Discovery and Development, through laboratory COMPUTATIONAL BIOLOGY

Qiu et al. PNAS | December 18, 2018 | vol. 115 | no. 51 | 12989 Downloaded by guest on September 29, 2021 space and facilities. This research was also supported, in part, by the Howard by NIH Grants GM103310 and S10 OD019994-01 and the Simons Foundation Hughes Medical Institute and NIH Grant R01 GM29169 (to J.F.), by Public (349247) to Bridget Carragher and Clint Potter; and Ravi C. Kalathur, Renato Health Service Grants DA024022 and DA044855 (to Y.Z.), and by NIH Grants Bruni, Brian Kloss, and Filippo Mancia for constructive comments and advice R01 GM107462 and P41 GM116799 (to W.A.H.). We thank Klaas Martinus Pos on nanoparticle systems from the Center on Membrane Protein Production for the AcrB expression plasmid; Bill Rice and Ed Eng for help in data collection and Analysis, which is supported at the New York Structural Biology Center by on Titan Krios #2 at the Simons Electron Microscopy Center, which is supported NIH Grant P41 GM116799.

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