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Structure and Activity of Lipid Bilayer Within a Membrane-Protein Transporter

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Structure and Activity of Lipid Bilayer Within a Membrane-Protein Transporter Structure and activity of lipid bilayer within a membrane-protein 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 Biology, 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 Biochemistry 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 proteins function in native cell membranes, but extrac- 1.9 Å (30). Nevertheless, the mechanism of active transport 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 lipid bilayer may exist (26). ticles to study the multidrug exporter AcrB, by cryo-EM at 3.2-Å Although detergent molecules and some alkane 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 | phospholipid | styrene maleic acid 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 organisms, 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 lipids 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 lipoprotein 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 Electron 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 cell membrane 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 mole- 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), carbon (gray), and nitrogen (blue).
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