Abstract
G protein-coupled receptors (GPCRs) constitute a highly diverse and ubiquitous family of integral membrane proteins, transmitting signals inside the cells in response to an assortment of disparate extracellular stimuli. Their strategic location on the cell surface and their involvement in crucial cellular and physiological processes turn these receptors into highly important pharmaceutical targets. Recent technological developments aimed at stabilization and crystallization of these receptors have led to significant breakthroughs in GPCR structure determination efforts. One of the successful approaches involved receptor stabilization with the help of a fusion partner combined with crystallization in lipidic cubic phase (LCP). The success of using an LCP matrix for crystallization is generally attributed to the creation of a more native, membrane-like stabilizing environment for GPCRs just prior to nucleation and to the formation of type I crystal lattices, thus generating highly ordered and strongly diffracting crystals. Here we describe protocols for reconstituting purified GPCRs in LCP, performing pre-crystallization assays, setting up crystallization trials in manual mode, detecting crystallization hits, optimizing crystallization conditions, harvesting, and collecting crystallographic data The protocols provide a sensible framework for approaching crystallization of stabilized GPCRs in LCP, however, as in any crystallization experiment, extensive screening and optimization of crystallization conditions as well as optimization of protein construct and purification steps are required. The process remains risky and these protocols do not necessarily guarantee success.
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References
Fredriksson R, Lagerstrom MC, Lundin LG, Schioth HB (2003) The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol 63:1256–1272
Rubenstein K (2008) GPCRs: dawn of a new era? Cambridge Healthtech Institute, Needham
Jacoby E, Bouhelal R, Gerspacher M, Seuwen K (2006) The 7 TM G-protein-coupled receptor target family. ChemMedChem 1:761–782
Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M (2000) Crystal structure of rhodopsin: A G protein-coupled receptor. Science 289:739–745
Bosier B, Hermans E (2007) Versatility of GPCR recognition by drugs: from biological implications to therapeutic relevance. Trends Pharmacol Sci 228:438–446
Day PW, Rasmussen SG, Parnot C, Fung JJ, Masood A, Kobilka TS, Yao XJ, Choi HJ, Weis WI, Rohrer DK, Kobilka BK (2007) A monoclonal antibody for G protein-coupled receptor crystallography. Nat Methods 4:927–929
Rasmussen SG, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, Burghammer M, Ratnala VR, Sanishvili R, Fischetti RF, Schertler GF, Weis WI, Kobilka BK (2007) Crystal structure of the human beta2 adrenergic G-protein-coupled receptor. Nature 450:383–387
Rosenbaum DM, Cherezov V, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Yao XJ, Weis WI, Stevens RC, Kobilka BK (2007) GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 318:1266–1273
Cherezov V, Rosenbaum DM, Hanson MA, Rasmussen SG, Thian FS, Kobilka TS, Choi HJ, Kuhn P, Weis WI, Kobilka BK, Stevens RC (2007) High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 318:1258–1265
Jaakola VP, Griffith MT, Hanson MA, Cherezov V, Chien EY, Lane JR, Ijzerman AP, Stevens RC (2008) The 2.6 Angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322:1211–1217
Serrano-Vega MJ, Magnani F, Shibata Y, Tate CG (2008) Conformational thermostabilization of the beta1-adrenergic receptor in a detergent-resistant form. Proc Natl Acad Sci U S A 105:877–882
Magnani F, Shibata Y, Serrano-Vega MJ, Tate CG (2008) Co-evolving stability and conformational homogeneity of the human adenosine A2a receptor. Proc Natl Acad Sci U S A 105:10744–10749
Warne T, Serrano-Vega MJ, Baker JG, Moukhametzianov R, Edwards PC, Henderson R, Leslie AG, Tate CG, Schertler GF (2008) Structure of a beta1-adrenergic G-protein-coupled receptor. Nature 454:486–491
Hanson MA, Cherezov V, Griffith MT, Roth CB, Jaakola VP, Chien EY, Velasquez J, Kuhn P, Stevens RC (2008) A specific cholesterol binding site is established by the 2.8 A structure of the human beta(2)-adrenergic receptor. Structure 16:897–905
Katritch V, Cherezov V, Hanson MA, Roth RB, Abagyan R (2009) Analysis of the β2AR structure provides insight into agonist binding and role of the TM5 helix in the activation mechanism. J Mol Recognit (in press)
Mustafi D, Palczewski K (2009) Topology of class A G protein-coupled receptors: insights gained from crystal structures of rhodopsins, adrenergic and adenosine receptors. Mol Pharmacol 75:1–12
Reynolds KA, Katritch V, Abagyan R (2009) Identifying conformational changes of the beta(2) adrenoceptor that enable accurate prediction of ligand/receptor interactions and screening for GPCR modulators. J Comput Aided Mol Des (in press)
Scheerer P, Park JH, Hildebrand PW, Kim YJ, Krauss N, Choe HW, Hofmann KP, Ernst OP (2009) Crystal structure of opsin in its G-protein-interacting conformation. Nature 455:497–502
Caffrey M (2009) Crystallizing membrane proteins for structure determination: use of lipidic mesophases. Annu Rev Biophys 38:29–51
Caffrey M, Cherezov V (2009) Crystallizing membrane proteins using lipidic mesophases. Nat Protocols (in press)
Cheng A, Hummel B, Qiu H, Caffrey M (1998) A simple mechanical mixer for small viscous lipid-containing samples. Chem Phys Lipids 95:11–21
Cherezov V, Clogston J, Misquitta Y, Abdel-Gawad W, Caffrey M (2002) Membrane protein crystallization in meso: lipid type-tailoring of the cubic phase. Biophys J 83:3393–3407
Cherezov V, Liu J, Hanson MA, Griffith MT, Stevens RC (2008) LCP-FRAP assay for pre-screening membrane proteins for in meso crystallization. J Cryst Growth Design 8:4307–4315
Cherezov V, Peddi A, Muthusubramaniam L, Zheng YF, Caffrey M (2004) A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases. Acta Crystallogr D Biol Crystallogr 60:1795–1807
Soumpasis DM (1983) Theoretical analysis of fluorescence photobleaching recovery experiments. Biophys J 41:95–97
Cherezov V, Caffrey M (2007) Miniaturization and automation for high-throughput membrane protein crystallization in lipidic mesophases. In: Chayen NE (ed) Protein crystallization strategies for structural genomics. San Diego, International University Line
Cherezov V, Caffrey M (2003) Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins. J Appl Crystallogr 36:1372–1377
Lunde CS, Rouhani S, Facciotti MT, Glaeser RM (2006) Membrane-protein stability in a phospholipid-based crystallization medium. J Struct Biol 154:223–231
Forsythe E, Achari A, Pusey ML (2006) Trace fluorescent labeling for high-throughput crystallography. Acta Crystallogr D Biol Crystallogr 62:339–346
Cherezov V, Fersi H, Caffrey M (2001) Crystallization screens: compatibility with the lipidic cubic phase for in meso crystallization of membrane proteins. Biophys J 81:225–242
Vargas R, Mateu L, Romero R (2003) The effect of increasing concentrations of precipitating salts used to crystallize proteins on the structure of the lipidic Q224 cubic phase. Chem Phys Lipids 127:103–111
Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326
Kabsch W (1993) Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J Appl Crystallogr 26:795–800
Landau EM, Rosenbusch JP (1996) Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc Natl Acad Sci U S A 93:14532–14535
Misquitta Y, Caffrey M (2003) Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization. Biophys J 85:3084–3096
Ai X, Caffrey M (2000) Membrane protein crystallization in lipidic mesophases: detergent effects. Biophys J 79:394–405
Cherezov V, Caffrey M (2005) A simple and inexpensive nanoliter-volume dispenser for highly viscous materials used in membrane protein crystallization. J Appl Crystallogr 38:398–400
Qiu H, Caffrey M (2000) The phase diagram of the monoolein/water system: metastability and equilibrium aspects. Biomaterials 21:223–234
Misquitta Y, Cherezov V, Havas F, Patterson S, Mohan JM, Wells AJ, Hart DJ, Caffrey M (2004) Rational design of lipid for membrane protein crystallization. J Struct Biol 148:169–175
Yamashita J, Shiono M, Hato M (2008) New lipid family that forms inverted cubic phases in equilibrium with excess water: molecular structure–aqueous phase structure relationship for lipids with 5, 9, 13, 17-tetramethyloctadecyl and 5, 9, 13, 17-tetramethyloctadecanoyl chains. J Phys Chem B 112:12286–12296
Cherezov V, Clogston J, Papiz MZ, Caffrey M (2006) Room to move: crystallizing membrane proteins in swollen lipidic mesophases. J Mol Biol 357:1605–1618
Nollert P, Landau EM (1998) Enzymic release of crystals from lipidic cubic phases. Biochem Soc Trans 26:709–713
Luecke H, Schobert B, Richter HT, Cartailler JP, Lanyi JK (1999) Structure of bacteriorhodopsin at 1.55 A resolution. J Mol Biol 291:899–911
Acknowledgements
This work was supported in part by the NIH Roadmap Initiative grant P50 GM073197 (JCIMPT) and the Protein Structure Initiative grant U54 GM074961 (ATCG3D). The authors acknowledge contributions from colleagues Michael A. Hanson, Wei Liu, Jeffrey Liu, Mark Griffith, Ellen Chien, Veli-Pekka Jaakola, Chris Roth and Peter Kuhn.
The authors acknowledge the support of Janet Smith, Robert Fischetti, and the GM/CA-CAT team at the Advanced Photon Source, for assistance in development and use of the minibeam and beamtime. The GM/CA-CAT beamline (23-ID) is supported by the National Cancer Institute (Y1-CO-1020) and the National Institute of General Medical Sciences (Y1-GM-1104).
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Cherezov, V., Abola, E., Stevens, R.C. (2010). Recent Progress in the Structure Determination of GPCRs, a Membrane Protein Family with High Potential as Pharmaceutical Targets. In: Lacapère, JJ. (eds) Membrane Protein Structure Determination. Methods in Molecular Biology, vol 654. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-762-4_8
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DOI: https://doi.org/10.1007/978-1-60761-762-4_8
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