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A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies

Abstract

The thermostability of an integral membrane protein (MP) in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals that are suitable for structure determination. However, many mammalian MPs are too unstable for crystallization. We developed a thermostabilization strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes 6–12 months to thermostabilize a G-protein-coupled receptor (GPCR) containing 300 amino acid (aa) residues. The resulting thermostabilized MPs are more easily crystallized and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs because it is possible to determine multiple structures of the thermostabilized receptors bound to low-affinity ligands. Protocols and advice are given on how to develop thermostability assays for MPs and how to combine mutations to make an optimally stable mutant suitable for structural studies. The steps in the procedure include the generation of 300 site-directed mutants by Ala/Leu scanning mutagenesis, the expression of each mutant in mammalian cells by transient transfection and the identification of thermostable mutants using a thermostability assay that is based on binding of an 125I-labeled radioligand to the unpurified, detergent-solubilized MP. Individual thermostabilizing point mutations are then combined to make an optimally stable MP that is suitable for structural biology and other biophysical studies.

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Figure 1: Flowchart illustrating the thermostabilization strategy.
Figure 2: Optimization of transient transfection in HEK293 cells.
Figure 3: Different formats of the thermostability assays.
Figure 4: Development of a thermostability assay for the serotonin transporter.
Figure 5: Thermostabilization of the β1-adrenergic receptor.
Figure 6: Thermostabilization of the adenosine A2A receptor in the agonist-bound conformation.
Figure 7: Thermostability of the ultrastable β1AR mutant, JM50.

References

  1. 1

    Bill, R.M. et al. Overcoming barriers to membrane protein structure determination. Nat. Biotechnol. 29, 335–340 (2011).

    Article  CAS  Google Scholar 

  2. 2

    Drew, D., Lerch, M., Kunji, E., Slotboom, D.J. & de Gier, J.W. Optimization of membrane protein overexpression and purification using GFP fusions. Nat. Methods 3, 303–313 (2006).

    Article  CAS  Google Scholar 

  3. 3

    Drew, D. et al. A scalable, GFP-based pipeline for membrane protein overexpression screening and purification. Protein Sci. 14, 2011–2017 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Hattori, M., Hibbs, R.E. & Gouaux, E. A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure 20, 1293–1299 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).

    Article  CAS  Google Scholar 

  6. 6

    Mancia, F. & Love, J. High throughput platforms for structural genomics of integral membrane proteins. Curr. Opin. Struct. Biol. 21, 517–522 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Drew, D. et al. GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae. Nat. Protoc. 3, 784–798 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Newstead, S., Kim, H., von Heijne, G., Iwata, S. & Drew, D. High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 104, 13936–13941 (2007).

    Article  CAS  Google Scholar 

  9. 9

    Sonoda, Y. et al. Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures. Structure 19, 17–25 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Zhang, X., Stevens, R.C. & Xu, F. The importance of ligands for G protein-coupled receptor stability. Trends Biochem. Sci. 40, 79–87 (2015).

    Article  CAS  Google Scholar 

  11. 11

    Tate, C.G. A crystal clear solution for determining G-protein-coupled receptor structures. Trends Biochem. Sci. 37, 343–352 (2012).

    Article  CAS  Google Scholar 

  12. 12

    Magnani, F., Shibata, Y., Serrano-Vega, M.J. & Tate, C.G. Co-evolving stability and conformational homogeneity of the human adenosine A2a receptor. Proc. Natl. Acad. Sci. USA 105, 10744–10749 (2008).

    Article  Google Scholar 

  13. 13

    Serrano-Vega, M.J., Magnani, F., Shibata, Y. & Tate, C.G. Conformational thermostabilization of the β1-adrenergic receptor in a detergent-resistant form. Proc. Natl. Acad. Sci. USA 105, 877–882 (2008).

    Article  Google Scholar 

  14. 14

    Shibata, Y. et al. Thermostabilization of the neurotensin receptor NTS1. J. Mol. Biol. 390, 262–277 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Sato, T. et al. Understanding the activation of the β1-adrenergic receptor through pharmacological analysis of 7-methylcyanopindolol and structure determination of the receptor-ligand complex. Mol. Pharm. 88, 1024–1034 (2015).

    Article  CAS  Google Scholar 

  16. 16

    Warne, T. et al. The structural basis for agonist and partial agonist action on a β(1)-adrenergic receptor. Nature 469, 241–244 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Christopher, J.A. et al. Biophysical fragment screening of the β1-adrenergic receptor: identification of high affinity arylpiperazine leads using structure-based drug design. J. Med. Chem. 56, 3446–3455 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Miller-Gallacher, J.L. et al. The 2.1 Å resolution structure of cyanopindolol-bound β1-adrenoceptor identifies an intramembrane Na+ ion that stabilises the ligand-free receptor. PLoS ONE 9, e92727 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Moukhametzianov, R. et al. Two distinct conformations of helix 6 observed in antagonist-bound structures of a β1-adrenergic receptor. Proc. Natl. Acad. Sci. USA 108, 8228–8232 (2011).

    Article  Google Scholar 

  20. 20

    Warne, T. et al. Structure of a β1-adrenergic G-protein-coupled receptor. Nature 454, 486–491 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Warne, T., Edwards, P.C., Leslie, A.G. & Tate, C.G. Crystal structures of a stabilized β1-adrenoceptor bound to the biased agonists bucindolol and carvedilol. Structure 20, 841–849 (2012).

    Article  CAS  Google Scholar 

  22. 22

    Lebon, G., Edwards, P.C., Leslie, A.G. & Tate, C.G. Molecular determinants of CGS21680 binding to the human adenosine A2A receptor. Mol. Pharmacol. 87, 907–915 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Lebon, G. et al. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 474, 521–525 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Congreve, M. et al. Discovery of 1,2,4-triazine derivatives as adenosine A(2A) antagonists using structure based drug design. J. Med. Chem. 55, 1898–1903 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Dore, A.S. et al. Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine. Structure 19, 1283–1293 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    White, J.F. et al. Structure of the agonist-bound neurotensin receptor. Nature 490, 508–513 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Egloff, P. et al. Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli. Proc. Natl. Acad. Sci. USA 111, E655–E662 (2014).

    Article  CAS  Google Scholar 

  28. 28

    Krumm, B.E., White, J.F., Shah, P. & Grisshammer, R. Structural prerequisites for G-protein activation by the neurotensin receptor. Nat. Commun. 6, 7895 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Leslie, A.G., Warne, T. & Tate, C.G. Ligand occupancy in crystal structure of β1-adrenergic G protein-coupled receptor. Nat. Struct. Mol. Biol. 22, 941–942 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Robertson, N. et al. The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery. Neuropharmacology 60, 36–44 (2011).

    Article  CAS  Google Scholar 

  31. 31

    Hollenstein, K. et al. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 499, 438–443 (2013).

    Article  CAS  Google Scholar 

  32. 32

    Dore, A.S. et al. Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain. Nature 511, 557–562 (2014).

    Article  CAS  Google Scholar 

  33. 33

    Tan, Q. et al. Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex. Science 341, 1387–1390 (2013).

    Article  CAS  Google Scholar 

  34. 34

    Srivastava, A. et al. High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875. Nature 513, 124–127 (2014).

    Article  CAS  Google Scholar 

  35. 35

    Penmatsa, A., Wang, K.H. & Gouaux, E. X-ray structure of dopamine transporter elucidates antidepressant mechanism. Nature 503, 85–90 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Chen, L., Durr, K.L. & Gouaux, E. X-ray structures of AMPA receptor-cone snail toxin complexes illuminate activation mechanism. Science 345, 1021–1026 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Tucker, J. & Grisshammer, R. Purification of a rat neurotensin receptor expressed in Escherichia coli. Biochem. J. 317, 891–899 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Warne, T., Chirnside, J. & Schertler, G.F. Expression and purification of truncated, non-glycosylated turkey beta-adrenergic receptors for crystallization. Biochim. Biophys. Acta 1610, 133–140 (2003).

    Article  CAS  Google Scholar 

  39. 39

    Weiss, H.M. & Grisshammer, R. Purification and characterization of the human adenosine A(2a) receptor functionally expressed in Escherichia coli. Eur. J. Biochem. 269, 82–92 (2002).

    Article  CAS  Google Scholar 

  40. 40

    Lebon, G., Bennett, K., Jazayeri, A. & Tate, C.G. Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor. J. Mol. Biol. 409, 298–310 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Miller, J.L. & Tate, C.G. Engineering an ultra-thermostable β(1)-adrenoceptor. J. Mol. Biol. 413, 628–638 (2011).

    Article  CAS  Google Scholar 

  42. 42

    Serrano-Vega, M.J. & Tate, C.G. Transferability of thermostabilizing mutations between beta-adrenergic receptors. Mol. Membr. Biol. 26, 385–396 (2009).

    Article  CAS  Google Scholar 

  43. 43

    Shibata, Y. et al. Optimising the combination of thermostabilising mutations in the neurotensin receptor for structure determination. Biochim. Biophys. Acta 1828, 1293–1301 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Abdul-Hussein, S., Andrell, J. & Tate, C.G. Thermostabilisation of the serotonin transporter in a cocaine-bound conformation. J. Mol. Biol. 425, 2198–2207 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    AbdulHussein, S. Conformational thermostabilisation of a mammalian serotonin transporter. PhD thesis, University of Cambridge, UK, (2011).

  46. 46

    Andrell, J. & Tate, C.G. Overexpression of membrane proteins in mammalian cells for structural studies. Mol. Membr. Biol. 30, 52–63 (2013).

    Article  Google Scholar 

  47. 47

    Grisshammer, R. & Tate, C.G. Overexpression of integral membrane proteins for structural studies. Q. Rev. Biophys. 28, 315–422 (1995).

    Article  CAS  Google Scholar 

  48. 48

    Tate, C.G. Overexpression of mammalian integral membrane proteins for structural studies. FEBS Lett. 504, 94–98 (2001).

    Article  CAS  Google Scholar 

  49. 49

    Thomas, J. & Tate, C.G. Quality control in eukaryotic membrane protein overproduction. J. Mol. Biol. 426, 4139–4154 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Tate, C.G. et al. Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter. Biochim. Biophys. Acta 1610, 141–153 (2003).

    Article  CAS  Google Scholar 

  51. 51

    Tate, C.G. & Blakely, R.D. The effect of N-linked glycosylation on activity of the Na(+)- and Cl(-)-dependent serotonin transporter expressed using recombinant baculovirus in insect cells. J. Biol. Chem. 269, 26303–26310 (1994).

    CAS  PubMed  Google Scholar 

  52. 52

    Tate, C.G., Whiteley, E. & Betenbaugh, M.J. Molecular chaperones stimulate the functional expression of the cocaine-sensitive serotonin transporter. J. Biol. Chem. 274, 17551–17558 (1999).

    Article  CAS  Google Scholar 

  53. 53

    Tate, C.G. Practical considerations of membrane protein instability during purification and crystallisation. Methods Mol. Biol. 601, 187–203 (2010).

    Article  CAS  Google Scholar 

  54. 54

    Helenius, A. & Simons, K. Solubilization of membranes by detergents. Biochim. Biophys. Acta 415, 29–79 (1975).

    Article  CAS  Google Scholar 

  55. 55

    le Maire, M., Champeil, P. & Moller, J.V. Interaction of membrane proteins and lipids with solubilizing detergents. Biochim. Biophys. Acta 1508, 86–111 (2000).

    Article  CAS  Google Scholar 

  56. 56

    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).

    Article  CAS  Google Scholar 

  57. 57

    Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405, 647–655 (2000).

    Article  CAS  Google Scholar 

  58. 58

    Chae, P.S. et al. A new class of amphiphiles bearing rigid hydrophobic groups for solubilization and stabilization of membrane proteins. Chemistry 18, 9485–9490 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Butler, P.J., Ubarretxena-Belandia, I., Warne, T. & Tate, C.G. The Escherichia coli multidrug transporter EmrE is a dimer in the detergent-solubilised state. J. Mol. Biol. 340, 797–808 (2004).

    Article  CAS  Google Scholar 

  60. 60

    Warne, T., Serrano-Vega, M.J., Tate, C.G. & Schertler, G.F. Development and crystallization of a minimal thermostabilised G protein-coupled receptor. Protein Expr. Purif. 65, 204–213 (2009).

    Article  CAS  Google Scholar 

  61. 61

    Zhukov, A. et al. Biophysical mapping of the adenosine A2A receptor. J. Med. Chem. 54, 4312–4323 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. 62

    Rosenbaum, D.M. et al. GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function. Science 318, 1266–1273 (2007).

    Article  CAS  Google Scholar 

  63. 63

    Steyaert, J. & Kobilka, B.K. Nanobody stabilization of G protein-coupled receptor conformational states. Curr. Opin. Struct. Biol. 21, 567–572 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

    Caffrey, M. Crystallizing membrane proteins for structure determination: use of lipidic mesophases. Annu. Rev. Biophys. 38, 29–51 (2009).

    Article  CAS  Google Scholar 

  65. 65

    Zhou, Y. & Bowie, J.U. Building a thermostable membrane protein. J. Biol. Chem. 275, 6975–6979 (2000).

    Article  CAS  Google Scholar 

  66. 66

    Li, D. et al. Crystal structure of the integral membrane diacylglycerol kinase. Nature 497, 521–524 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. 67

    Grisshammer, R., Duckworth, R. & Henderson, R. Expression of a rat neurotensin receptor in Escherichia coli. Biochem. J. 295, 571–576 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. 68

    Sarkar, C.A. et al. Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity. Proc. Natl. Acad. Sci. USA 105, 14808–14813 (2008).

    Article  Google Scholar 

  69. 69

    Scott, D.J. & Pluckthun, A. Direct molecular evolution of detergent-stable G protein-coupled receptors using polymer encapsulated cells. J. Mol. Biol. 425, 662–677 (2013).

    Article  CAS  Google Scholar 

  70. 70

    Isogai, S. et al. Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor. Nature 530, 237–241 (2016).

    Article  CAS  Google Scholar 

  71. 71

    Zhao, J., Benlekbir, S. & Rubinstein, J.L. Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Nature 521, 241–245 (2015).

    Article  CAS  Google Scholar 

  72. 72

    Allegretti, M. et al. Horizontal membrane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase. Nature 521, 237–240 (2015).

    Article  CAS  Google Scholar 

  73. 73

    Congreve, M., Langmead, C.J., Mason, J.S. & Marshall, F.H. Progress in structure based drug design for G protein-coupled receptors. J. Med. Chem. 54, 4283–4311 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. 74

    Jazayeri, A., Dias, J.M. & Marshall, F.H. From G protein-coupled receptor structure resolution to rational drug design. J. Biol. Chem. 290, 19489–19495 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. 75

    Bennett, K.A. et al. Pharmacology and structure of isolated conformations of the adenosine A(2)A receptor define ligand efficacy. Mol. Pharmacol. 83, 949–958 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. 76

    Zheng, L., Baumann, U. & Reymond, J.L. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 32, e115 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  77. 77

    Palczewski, K. et al. Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. 78

    Tate, C.G. & Schertler, G.F. Engineering G protein-coupled receptors to facilitate their structure determination. Curr. Opin. Struct. Biol. 19, 386–395 (2009).

    Article  CAS  Google Scholar 

  79. 79

    Alexandrov, A.I., Mileni, M., Chien, E.Y., Hanson, M.A. & Stevens, R.C. Microscale fluorescent thermal stability assay for membrane proteins. Structure 16, 351–359 (2008).

    Article  CAS  Google Scholar 

  80. 80

    Dodevski, I. & Pluckthun, A. Evolution of three human GPCRs for higher expression and stability. J. Mol. Biol. 408, 599–615 (2011).

    Article  CAS  Google Scholar 

  81. 81

    Schlinkmann, K.M. et al. Maximizing detergent stability and functional expression of a GPCR by exhaustive recombination and evolution. J. Mol. Biol. 422, 414–428 (2012).

    Article  CAS  Google Scholar 

  82. 82

    Schaffner, W. & Weissmann, C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal. Biochem. 56, 502–514 (1973).

    Article  CAS  Google Scholar 

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Acknowledgements

Funding for the thermostabilization of membrane proteins in the laboratory of C.G.T. was from the Medical Research Council (MRC U105197215), Medical Research Council Technology Development Gap Fund, Pfizer, Heptares Therapeutics and an ERC Advanced Grant (EMPSI 339995). We thank R. Henderson, F. Marshall, A. Jazayeri and M. Weir for constructive comments on the manuscript.

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All authors contributed to the development of techniques described in this paper. C.G.T. wrote the manuscript and coordinated contributions from all the other authors.

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Correspondence to Christopher G Tate.

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C.G.T. is a consultant for Heptares Therapeutics, and this work was funded partly by Pfizer and Heptares Therapeutics.

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Magnani, F., Serrano-Vega, M., Shibata, Y. et al. A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies. Nat Protoc 11, 1554–1571 (2016). https://doi.org/10.1038/nprot.2016.088

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