Abstract
Within cell membranes numerous protein assemblies reside. Among their many functions, these assemblies regulate the movement of molecules between membranes, facilitate signaling into and out of cells, allow movement of cells by cell-matrix attachment, and regulate the electric potential of the membrane. With such critical roles, membrane protein complexes are of considerable interest for human health, yet they pose an enduring challenge for structural biologists because it is difficult to study these protein structures at atomic resolution in in situ environments. To advance structural and functional insights for these protein assemblies, membrane mimetics are typically employed to recapitulate some of the physical and chemical properties of the lipid bilayer membrane. However, extraction from native membranes can sometimes change the structure and lipid-binding properties of these complexes, leading to conflicting results and fueling a drive to study complexes directly from native membranes. Here we consider the co-development of membrane mimetics with technological breakthroughs in both cryo-electron microscopy (cryo-EM) and native mass spectrometry (nMS). Together, these developments are leading to a plethora of high-resolution protein structures, as well as new knowledge of their lipid interactions, from different membrane-like environments.
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Callaway, E. The revolution will not be crystallized: a new method sweeps through structural biology. Nature 525, 172–174 (2015).
Davies, K. M., Anselmi, C., Wittig, I., Faraldo-Gómez, J. D. & Kühlbrandt, W. Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae. Proc. Natl. Acad. Sci. USA 109, 13602–13607 (2012).
Baker, L. A., Watt, I. N., Runswick, M. J., Walker, J. E. & Rubinstein, J. L. Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM. Proc. Natl. Acad. Sci. USA 109, 11675–11680 (2012).
Murphy, B. J. et al. Rotary substates of mitochondrial ATP synthase reveal the basis of flexible F1-Fo coupling. Science 364, eaaw9128 (2019).
Gu, J. et al. Cryo-EM structure of the mammalian ATP synthase tetramer bound with inhibitory protein IF1. Science 364, 1068–1075 (2019).
Spikes, T. E., Montgomery, M. G. & Walker, J. E. Structure of the dimeric ATP synthase from bovine mitochondria. Proc. Natl Acad. Sci. USA 117, 23519–23526 (2020).
Pinke, G., Zhou, L. & Sazanov, L. A. Cryo-EM structure of the entire mammalian F-type ATP synthase. Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-020-0503-8 (2020).
Seddon, A. M., Curnow, P. & Booth, P. J. Membrane proteins, lipids and detergents: not just a soap opera. Biochim. Biophys. Acta 1666, 105–117 (2004).
Burgess, N. K., Stanley, A. M. & Fleming, K. G. Determination of membrane protein molecular weights and association equilibrium constants using sedimentation equilibrium and sedimentation velocity. Methods Cell Biol. 84, 181–211 (2008).
Vukoti, K., Kimura, T., Macke, L., Gawrisch, K. & Yeliseev, A. Stabilization of functional recombinant cannabinoid receptor CB(2) in detergent micelles and lipid bilayers. PLoS One 7, e46290 (2012).
Barrera, N. P., Di Bartolo, N., Booth, P. J. & Robinson, C. V. Micelles protect membrane complexes from solution to vacuum. Science 321, 243–246 (2008). This is a report of a membrane protein complex ejected from a detergent micelle into the gas phase of a mass spectrometer with cytoplasmic and membrane domains intact.
Reading, E. et al. The role of the detergent micelle in preserving the structure of membrane proteins in the gas phase. Angew. Chem. Int. Edn Engl. 54, 4577–4581 (2015).
Liko, I. et al. Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding. Proc. Natl. Acad. Sci. USA 113, 8230–8235 (2016).
Zhou, M. et al. Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding. Science 334, 380–385 (2011).
Sušac, L., Eddy, M. T., Didenko, T., Stevens, R. C. & Wüthrich, K. A2A adenosine receptor functional states characterized by 19F-NMR. Proc. Natl. Acad. Sci. USA 115, 12733–12738 (2018).
Palczewski, K. et al. Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289, 739–745 (2000). This study reports the structure of an unmodified wild-type GPCR, allowing insights into the propagation of signals in vision.
Cherezov, V. et al. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007).
Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 318, 1266–1273 (2007).
Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011).
Chae, P. S. et al. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nat. Methods 7, 1003–1008 (2010).
Caffrey, M. A comprehensive review of the lipid cubic phase or in meso method for crystallizing membrane and soluble proteins and complexes. Acta Crystallogr. F Struct. Biol. Commun. 71, 3–18 (2015).
Yen, H. Y. et al. PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling. Nature 559, 423–427 (2018). This study reports the use of mass spectrometry to uncover the role of PIP2 in stabilizing downstream coupling of class A GPCRs.
Huang, W. et al. Structure of the neurotensin receptor 1 in complex with β-arrestin 1. Nature 579, 303–308 (2020). This paper reports the cryo-EM structure of an arrestin-bound receptor, also revealing a PIP2 molecule forming a bridge between the membrane side of the receptor and arrestin.
Shen, H., Liu, D., Wu, K., Lei, J. & Yan, N. Structures of human Nav1.7 channel in complex with auxiliary subunits and animal toxins. Science 363, 1303–1308 (2019).
Urner, L. H. et al. Modular detergents tailor the purification and structural analysis of membrane proteins including G-protein coupled receptors. Nat. Commun. 11, 564 (2020).
Dorwart, M. R., Wray, R., Brautigam, C. A., Jiang, Y. & Blount, P. S. aureus MscL is a pentamer in vivo but of variable stoichiometries in vitro: implications for detergent-solubilized membrane proteins. PLoS Biol. 8, e1000555 (2010).
Reading, E. et al. The effect of detergent, temperature, and lipid on the oligomeric state of MscL constructs: insights from mass spectrometry. Chem. Biol. 22, 593–603 (2015).
Chipot, C. et al. Perturbations of native membrane protein structure in alkyl phosphocholine detergents: a critical assessment of NMR and biophysical studies. Chem. Rev. 118, 3559–3607 (2018).
Lemieux, M. J., Reithmeier, R. A. & Wang, D. N. Importance of detergent and phospholipid in the crystallization of the human erythrocyte anion-exchanger membrane domain. J. Struct. Biol. 137, 322–332 (2002).
Drachmann, N. D. et al. Comparing crystal structures of Ca(2+) -ATPase in the presence of different lipids. FEBS J. 281, 4249–4262 (2014).
Laganowsky, A. et al. Membrane proteins bind lipids selectively to modulate their structure and function. Nature 510, 172–175 (2014).
Gupta, K. et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature 541, 421–424 (2017).
Sanders, M. R., Findlay, H. E. & Booth, P. J. Lipid bilayer composition modulates the unfolding free energy of a knotted α-helical membrane protein. Proc. Natl. Acad. Sci. USA 115, E1799–E1808 (2018).
Karabadzhak, A. G. et al. Bilayer thickness and curvature influence binding and insertion of a pHLIP peptide. Biophys. J. 114, 2107–2115 (2018).
Landreh, M., Costeira-Paulo, J., Gault, J., Marklund, E. G. & Robinson, C. V. Effects of detergent micelles on lipid binding to proteins in electrospray ionization mass spectrometry. Anal. Chem. 89, 7425–7430 (2017).
Zoonens, M., Catoire, L. J., Giusti, F. & Popot, J. L. NMR study of a membrane protein in detergent-free aqueous solution. Proc. Natl. Acad. Sci. USA 102, 8893–8898 (2005).
Calabrese, A. N., Watkinson, T. G., Henderson, P. J., Radford, S. E. & Ashcroft, A. E. Amphipols outperform dodecylmaltoside micelles in stabilizing membrane protein structure in the gas phase. Anal. Chem. 87, 1118–1126 (2015).
Chien, C. H. et al. An adaptable phospholipid membrane mimetic system for solution NMR studies of membrane proteins. J. Am. Chem. Soc. 139, 14829–14832 (2017).
Elter, S. et al. The use of amphipols for NMR structural characterization of 7-TM proteins. J. Membr. Biol. 247, 957–964 (2014).
Diver, M. M., Cheng, Y. & Julius, D. Structural insights into TRPM8 inhibition and desensitization. Science 365, 1434–1440 (2019).
McLean, M. A., Gregory, M. C. & Sligar, S. G. Nanodiscs: a controlled bilayer surface for the study of membrane proteins. Annu. Rev. Biophys. 47, 107–124 (2018).
Autzen, H. E., Julius, D. & Cheng, Y. Membrane mimetic systems in CryoEM: keeping membrane proteins in their native environment. Curr. Opin. Struct. Biol. 58, 259–268 (2019).
Yokogawa, M., Fukuda, M. & Osawa, M. Nanodiscs for structural biology in a membranous environment. Chem. Pharm. Bull. (Tokyo) 67, 321–326 (2019).
Gao, Y., Cao, E., Julius, D. & Cheng, Y. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534, 347–351 (2016).
Qiu, W. et al. Structure and activity of lipid bilayer within a membrane-protein transporter. Proc. Natl. Acad. Sci. USA 115, 12985–12990 (2018).
Postis, V. et al. The use of SMALPs as a novel membrane protein scaffold for structure study by negative stain electron microscopy. Biochim. Biophys. Acta 1848, 496–501 (2015).
Sun, C. et al. Structure of the alternative complex III in a supercomplex with cytochrome oxidase. Nature 557, 123–126 (2018).
Brady, N. G., Li, M., Ma, Y., Gumbart, J. C. & Bruce, B. D. Non-detergent isolation of a cyanobacterial photosystem I using styrene maleic acid alternating copolymers. RSC Advances 9, 31781–31796 (2019).
Hall, S. C. L. et al. An acid-compatible co-polymer for the solubilization of membranes and proteins into lipid bilayer-containing nanoparticles. Nanoscale 10, 10609–10619 (2018).
Ruan, J., Xia, S., Liu, X., Lieberman, J. & Wu, H. Cryo-EM structure of the gasdermin A3 membrane pore. Nature 557, 62–67 (2018).
Peng, W., de Souza Santos, M., Li, Y., Tomchick, D. R. & Orth, K. High-resolution cryo-EM structures of the E. coli hemolysin ClyA oligomers. PLoS One 14, e0213423 (2019).
Wang, L. & Sigworth, F. J. Structure of the BK potassium channel in a lipid membrane from electron cryomicroscopy. Nature 461, 292–295 (2009).
Jiang, Q. X., Chester, D. W. & Sigworth, F. J. Spherical reconstruction: a method for structure determination of membrane proteins from cryo-EM images. J. Struct. Biol. 133, 119–131 (2001).
Markones, M. et al. Stairway to asymmetry: five steps to lipid-asymmetric proteoliposomes. Biophys. J. 118, 294–302 (2020).
Carlson, M. L. et al. The Peptidisc, a simple method for stabilizing membrane proteins in detergent-free solution. eLife 7, e34085 (2018).
Frauenfeld, J. et al. A saposin-lipoprotein nanoparticle system for membrane proteins. Nat. Methods 13, 345–351 (2016).
Carlson, M. L. et al. Profiling the Escherichia coli membrane protein interactome captured in Peptidisc libraries. eLife 8, e46615 (2019).
Zeev-Ben-Mordehai, T., Vasishtan, D., Siebert, C. A., Whittle, C. & Grünewald, K. Extracellular vesicles: a platform for the structure determination of membrane proteins by Cryo-EM. Structure 22, 1687–1692 (2014).
Zeev-Ben-Mordehai, T., Vasishtan, D., Siebert, C. A. & Grünewald, K. The full-length cell-cell fusogen EFF-1 is monomeric and upright on the membrane. Nat. Commun. 5, 3912 (2014). This study reports the cryo-EM structure of a cell–cell fusogen imaged while in a membrane environment.
Chorev, D. S. et al. Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry. Science 362, 829–834 (2018). This paper reports a mass spectrometry study of membrane protein complexes ejected directly from their membrane environments including bacterial and mitochnodrial membranes.
Danev, R., Yanagisawa, H. & Kikkawa, M. Cryo-electron microscopy methodology: current aspects and future directions. Trends Biochem. Sci. 44, 837–848 (2019).
Laverty, D. et al. Cryo-EM structure of the human α1β3γ2 GABAA receptor in a lipid bilayer. Nature 565, 516–520 (2019).
Zhu, S. et al. Structure of a human synaptic GABAA receptor. Nature 559, 67–72 (2018).
Phulera, S. et al. Cryo-EM structure of the benzodiazepine-sensitive α1β1γ2S tri-heteromeric GABAA receptor in complex with GABA. eLife 7, e39383 (2018).
Rose, R. J., Damoc, E., Denisov, E., Makarov, A. & Heck, A. J. High-sensitivity Orbitrap mass analysis of intact macromolecular assemblies. Nat. Methods 9, 1084–1086 (2012). This study demonstrates a high-resolution Orbitrap for native MS of soluble protein complexes.
Gault, J. et al. High-resolution mass spectrometry of small molecules bound to membrane proteins. Nat. Methods 13, 333–336 (2016). This study covers the development of the Orbitrap platform for membrane proteins and demonstrates that the chain length of lipids could be distinguished while bound to the membrane protein.
Fort, K. L. et al. Expanding the structural analysis capabilities on an Orbitrap-based mass spectrometer for large macromolecular complexes. Analyst 143, 100–105 (2017).
Liu, Y. et al. Selective binding of a toxin and phosphatidylinositides to a mammalian potassium channel. Nat. Commun. 10, 1352 (2019).
Liko, I., Allison, T. M., Hopper, J. T. & Robinson, C. V. Mass spectrometry guided structural biology. Curr. Opin. Struct. Biol. 40, 136–144 (2016).
Abbas, Y. M., Wu, D., Bueler, S. A., Robinson, C. V. & Rubinstein, J. L. Structure of V-ATPase from the mammalian brain. Science 367, 1240–1246 (2020). This study reports the cryo-EM structure of the V-type ATPase isolated from rat brain synaptic vesicles. MS was used to define the isoform and subunit stoichiometry and composition of the intact V1 complex.
Hellwig, N. et al. Native mass spectrometry goes more native: investigation of membrane protein complexes directly from SMALPs. Chem. Commun. (Camb.) 54, 13702–13705 (2018).
Keener, J. E. et al. Chemical additives enable native mass spectrometry measurement of membrane protein oligomeric state within intact nanodiscs. J. Am. Chem. Soc. 141, 1054–1061 (2019).
Sousa, J. S., Mills, D. J., Vonck, J. & Kühlbrandt, W. Functional asymmetry and electron flow in the bovine respirasome. eLife 5, e21290 (2016).
Jussupow, A., Di Luca, A. & Kaila, V. R. I. How cardiolipin modulates the dynamics of respiratory complex I. Sci. Adv. 5, eaav1850 (2019).
Lu, F. et al. Structure and mechanism of the uracil transporter UraA. Nature 472, 243–246 (2011).
Yu, X. et al. Dimeric structure of the uracil:proton symporter UraA provides mechanistic insights into the SLC4/23/26 transporters. Cell Res. 27, 1020–1033 (2017).
Gong, X. et al. Structural basis for the recognition of Sonic Hedgehog by human Patched1. Science 361, eaas8935 (2018).
Zhang, Y. et al. Structural basis for cholesterol transport-like activity of the hedgehog receptor patched. Cell 175, 1352–1364.e14 (2018).
Qian, H. et al. Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm. Nat. Commun. 10, 2320 (2019).
Bakelar, J., Buchanan, S. K. & Noinaj, N. The structure of the β-barrel assembly machinery complex. Science 351, 180–186 (2016).
Han, L. et al. Structure of the BAM complex and its implications for biogenesis of outer-membrane proteins. Nat. Struct. Mol. Biol. 23, 192–196 (2016).
Iadanza, M. G. et al. Lateral opening in the intact β-barrel assembly machinery captured by cryo-EM. Nat. Commun. 7, 12865 (2016).
Celia, H. et al. Structural insight into the role of the Ton complex in energy transduction. Nature 538, 60–65 (2016).
Maki-Yonekura, S. et al. Hexameric and pentameric complexes of the ExbBD energizer in the Ton system. eLife 7, e35419 (2018).
Celia, H. et al. Cryo-EM structure of the bacterial Ton motor subcomplex ExbB-ExbD provides information on structure and stoichiometry. Commun Biol 2, 358 (2019).
Danev, R. & Baumeister, W. Expanding the boundaries of cryo-EM with phase plates. Curr. Opin. Struct. Biol. 46, 87–94 (2017).
Schwartz, O. et al. Laser phase plate for transmission electron microscopy. Nat. Methods 16, 1016–1020 (2019).
Zhou, A. et al. Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM. eLife 4, e10180 (2015).
Raschle, T., Hiller, S., Etzkorn, M. & Wagner, G. Nonmicellar systems for solution NMR spectroscopy of membrane proteins. Curr. Opin. Struct. Biol. 20, 471–479 (2010).
Acknowledgements
We thank all members of the Robinson group for helpful discussions and the many collaborators who have contributed to this work. We are also grateful to S.L. Rouse, L.A. Baker, N. Yan and C. Gerle for critical review of the manuscript. We would also like to thank F. Samsudin and S. Khalid for the MD based model of the Bam Complex. We acknowledge with thanks funding from an ERC Advanced Grant ENABLE (695511) and a Wellcome Trust Investigator Award (104633/Z/14/Z).
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Chorev, D.S., Robinson, C.V. The importance of the membrane for biophysical measurements. Nat Chem Biol 16, 1285–1292 (2020). https://doi.org/10.1038/s41589-020-0574-1
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DOI: https://doi.org/10.1038/s41589-020-0574-1
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