Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Small-scale approach for precrystallization screening in GPCR X-ray crystallography

Abstract

G protein–coupled receptors (GPCRs) are important pharmaceutical targets. Knowledge of their 3D structures is critical to understanding mechanisms of drug action. Low cellular expression, purification yield, and in vitro instability are substantial hurdles to the successful determination of GPCR structure. Intense effort is required to optimize a receptor’s protein sequence and purification procedure, increasing the complexity of the precrystallization process. Here, we present a procedure for a small-scale precrystallization screen that involves detecting GPCR expression levels in Spodoptera frugiperda (Sf9) culture by flow cytometry and evaluating GPCR stability by size-exclusion chromatography and UV absorbance measurements. The example procedure uses the smallest volumes of Sf9 cell culture that will yield sufficient quantities of purified protein for intrinsic UV absorbance analysis and is amenable to medium throughput with the same constructs and conditions that would be scaled up for crystallization trials. The protocol takes 8 d to complete and requires expertise in cell culture, protein purification, and chromatography.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Overview of the procedure.
Fig. 2: GPCR expression in small-scale (4-ml) Sf9 cell culture.
Fig. 3: Size-exclusion chromatography of purified small-scale culture expressing different EP3 receptor constructs.

Data availability

The datasets generated and analyzed here are available from the authors upon request.

References

  1. 1.

    Santos, R. et al. A comprehensive map of molecular drug targets. Nat. Rev. Drug Discov. 16, 19–34 (2016).

    Article  Google Scholar 

  2. 2.

    Sriram, K. & Insel, P. A. G protein-coupled receptors as targets for approved drugs: how many targets and how many drugs? Mol. Pharmacol. 93, 251–258 (2018).

    CAS  Article  Google Scholar 

  3. 3.

    Xiang, J. et al. Successful strategies to determine high-resolution structures of GPCRs. Trends Pharmacol. Sci. 37, 1055–1069 (2016).

    CAS  Article  Google Scholar 

  4. 4.

    Audet, M. & Stevens, R. C. Emerging structural biology of lipid G protein-coupled receptors. Protein Sci. 28, 292–304 (2019).

    CAS  Article  Google Scholar 

  5. 5.

    Audet, M. et al. Crystal structure of misoprostol bound to the labor inducer prostaglandin E2 receptor. Nat. Chem. Biol. 15, 11–17 (2019).

    CAS  Article  Google Scholar 

  6. 6.

    Li, X. et al. Crystal structure of the human cannabinoid receptor CB2. Cell 176, 459–467.e413 (2019).

    CAS  Article  Google Scholar 

  7. 7.

    Hua, T. et al. Crystal structures of agonist-bound human cannabinoid receptor CB1. Nature 547, 468–471 (2017).

    CAS  Article  Google Scholar 

  8. 8.

    Shao, Z. et al. High-resolution crystal structure of the human CB1 cannabinoid receptor. Nature 540, 602–606 (2016).

    CAS  Article  Google Scholar 

  9. 9.

    Hua, T. et al. Crystal structure of the human cannabinoid receptor CB1. Cell 167, 750–762.e714 (2016).

    CAS  Article  Google Scholar 

  10. 10.

    Wacker, D. et al. Crystal structure of an LSD-bound human serotonin receptor. Cell 168, 377–389.e312 (2017).

    CAS  Article  Google Scholar 

  11. 11.

    Kumar, A. & Plückthun, A. In vivo assembly and large-scale purification of a GPCR - Gα fusion with Gβγ, and characterization of the active complex. PloS one 14, e0210131 (2019).

    CAS  Article  Google Scholar 

  12. 12.

    Thal, D. M., Glukhova, A., Sexton, P. M. & Christopoulos, A. Structural insights into G-protein-coupled receptor allostery. Nature 559, 45–53 (2018).

    CAS  Article  Google Scholar 

  13. 13.

    Yang, S. et al. Crystal structure of the Frizzled 4 receptor in a ligand-free state. Nature 560, 666–670 (2018).

    CAS  Article  Google Scholar 

  14. 14.

    René, P. et al. Pharmacological chaperones restore function to MC4R mutants responsible for severe early-onset obesity. J. Pharmacol. Exp. Ther. 335, 520–532 (2010).

    Article  Google Scholar 

  15. 15.

    Morello, J. P. et al. Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants. J. Clin. Invest. 105, 887–895 (2000).

    CAS  Article  Google Scholar 

  16. 16.

    Lyons, J. A., Shahsavar, A., Paulsen, P. A., Pedersen, B. P. & Nissen, P. Expression strategies for structural studies of eukaryotic membrane proteins. Curr. Opin. Struct. Biol. 38, 137–144 (2016).

    CAS  Article  Google Scholar 

  17. 17.

    Elegheert, J. et al. Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins. Nat. Protoc. 13, 2991–3017 (2018).

    CAS  Article  Google Scholar 

  18. 18.

    Chen, H., Shaffer, P. L., Huang, X. & Rose, P. E. Rapid screening of membrane protein expression in transiently transfected insect cells. Protein Expr. Purif. 88, 134–142 (2013).

    CAS  Article  Google Scholar 

  19. 19.

    Suzuki, N. et al. An efficient screening method for purifying and crystallizing membrane proteins using modified clear-native PAGE. Anal. Biochem. 548, 7–14 (2018).

    CAS  Article  Google Scholar 

  20. 20.

    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).

    CAS  Article  Google Scholar 

  21. 21.

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

    CAS  Article  Google Scholar 

  22. 22.

    Shiroishi, M. et al. Platform for the rapid construction and evaluation of GPCRs for crystallography in Saccharomyces cerevisiae. Microb. Cell Fact. 11, 78–78 (2012).

    CAS  Article  Google Scholar 

  23. 23.

    Li, X. et al. Generation of destabilized green fluorescent protein as a transcription reporter. J. Biol. Chem. 273, 34970–34975 (1998).

    CAS  Article  Google Scholar 

  24. 24.

    Tsien, R. Y. The green fluorescent protein. Ann. Rev. Biochem. 67, 509–544 (1998).

    CAS  Article  Google Scholar 

  25. 25.

    Sokolovski, M., Bhattacherjee, A., Kessler, N., Levy, Y. & Horovitz, A. Thermodynamic protein destabilization by GFP tagging: a case of interdomain allostery. Biophys. J. 109, 1157–1162 (2015).

    CAS  Article  Google Scholar 

  26. 26.

    Rodriguez-Banqueri, A., Kowalczyk, L., Palacin, M. & Vazquez-Ibar, J. L. Assessment of membrane protein expression and stability using a split green fluorescent protein reporter. Anal. Biochem. 423, 7–14 (2012).

    CAS  Article  Google Scholar 

  27. 27.

    Toyoda, Y. et al. Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface. Nat. Chem. Biol. 15, 18–26 (2019).

    CAS  Article  Google Scholar 

  28. 28.

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

    CAS  Article  Google Scholar 

  29. 29.

    Liu, W. et al. Structural basis for allosteric regulation of GPCRs by sodium ions. Science 337, 232–236 (2012).

    CAS  Article  Google Scholar 

  30. 30.

    Gill, J. E., Jotz, M. M., Young, S. G., Modest, E. J. & Sengupta, S. K. 7-Amino-actinomycin D as a cytochemical probe. I. Spectral properties. J. Histochem. Cytochem. 23, 793–799 (1975).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This research was supported by NIH/NIGMS Protein Structure Initiative U54GM094618 (R.C.S.). M.A. was supported by a Canadian Institute of Health and Research (CIHR) Postdoctoral Fellowship Award. We thank A. Walker for assistance in manuscript preparation and F. Badeaux and E. Audet-Badeaux for their support.

Author information

Affiliations

Authors

Contributions

M.A. designed the study; cloned the constructs; and performed the baculovirus production, receptor expression, and receptor purification and characterization. J.V. cloned the constructs; K.V., M.C, and C.H. performed baculovirus production and receptor expression; M.A. and R.C.S. supervised the project.

Corresponding authors

Correspondence to Martin Audet or Raymond C. Stevens.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Gopala Krishna Aradhyam and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key reference using this protocol

Audet, M. et al. Nat. Chem. Biol. 15, 11–17 (2019): https://www.nature.com/articles/s41589-018-0160-y

Integrated supplementary information

Supplementary Fig. 1 Illustration of the gating strategy used in the flow cytometry experiment.

a, Plot of front scatter (FSC) against side scatter (SSC) signals shows that Sf9 insect cells growth as a homogeneous and monodispersed suspension culture. For GP64-PE and FLAG-FITC experiments, we measured 2000 events that show an FSC signal above 180 counts. We are not gating SSC signal. b, Typical GP64-PE experiment to monitor baculovirus budding on the surface of Sf9 cells after bacmid transfection. Positive GP64-PE signal is set above 20 counts. Non-transfected Sf9 cells are used as a negative control and shown in blue, Sf9 cells transfected with a bacmid containing the coding sequence of A2A-BRIL is shown in magenta. The gate was set at 20 counts for positive GP64-PE signal. c-d, Typical FLAG-FITC experiment to monitor c, cell surface and d, total receptor expression in Sf9 cells after baculovirus infection. Non-infected Sf9 cells are used as negative control and shown in blue. Sf9 cell expressing A2A-BRIL receptor fusion is shown in magenta. The gate was set at 30 counts for positive FLAG-FITC signal for both c, cell surface and d, total receptor expression. The gates are depicted on the graphs as solid lines.

Supplementary Fig. 2 Illustration of the EP3-rub sequence and truncations.

a, n- and b, c-terminal truncations of EP3-rub reference construct. The non-truncated EP3-rub is the isoform D of EP3 receptor fused with a rubredoxin flanked by a Gly-Ser linker on both sides (EP3-rub). The rubredoxin is inserted in the third intracellular loop of EP3 by replacing the receptor residues 260-267. A FLAG-tag and a 10 x his-tag were fused at both n- and c-terminus respectively. EP3 receptor residue number are indicated above each cartoon.

Supplementary information

Supplementary Information

Supplementary Figures 1 and 2 and Supplementary Table 1

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Audet, M., Villers, K., Velasquez, J. et al. Small-scale approach for precrystallization screening in GPCR X-ray crystallography. Nat Protoc 15, 144–160 (2020). https://doi.org/10.1038/s41596-019-0259-y

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing