Abstract
Structural studies of G-protein-coupled receptors (GPCRs) are often limited by difficulties in obtaining well-diffracting crystals suitable for high-resolution structure determination. During the past decade, crystallization in lipidic cubic phase (LCP) has become the most successful and widely used technique for obtaining such crystals. Despite often intense efforts, many GPCRs remain refractory to crystallization, even if receptors can be purified in sufficient amounts. To address this issue, we have developed a highly efficient screening and stabilization strategy for GPCRs, based on a fluorescence thermal stability assay readout, which seems to correlate particularly well with those GPCR constructs that remain native during incorporation into the LCP. Detailed protocols are provided for rapid and cost-efficient mutant and construct generation using sequence- and ligation-independent cloning, high-throughput magnetic bead-based protein purification from small-scale expressions in mammalian cells, the screening and optimal combination of mutations for increased receptor thermostability and the rapid identification of suitable chimeric fusion protein constructs for successful crystallization in LCP. We exemplify the method on three receptors from two different classes: the neurokinin 1 receptor, the oxytocin receptor and the parathyroid hormone 1 receptor.
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All data needed to evaluate the conclusions on the paper are present in the paper. The datasets generated and analyzed here are available from the authors upon request.
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Acknowledgements
We thank G. Meier for his help during transient transfection and expression of protein and would furthermore like to thank F. Zosel for critical reading of the manuscript. This work was supported by Schweizerischer Nationalfonds grant 31003A_153143 and 31003A_182334, and KTI grant 18022.1 PFLS-LS, all to A.P.
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J.S. conceptualized the project, devised and established the thermostabilization platform including the expression format and microscale purification technique, performed NK1R mutagenesis and thermostabilization, and designed and characterized NK1R crystallization constructs. J.E. devised and established the SLIC-based mutagenesis and construct generation platform and performed the PTH1R thermostabilization and crystallization construct screening. Y.W. performed the OTR thermostabilization and crystallization construct screening with help from J.S. The project was supervised by A.P. The manuscript was prepared by J.S., J.E., Y.W. and A.P. All authors contributed to the final editing and approved of the manuscript.
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Ehrenmann, J. et al. Nat. Struct. Mol. Biol. 25, 1086–1092 (2018): https://doi.org/10.1038/s41594-018-0151-4
Schöppe, J. et al. Nat. Commun. 10, 17 (2019): https://doi.org/10.1038/s41467-018-07939-8
Waltenspühl, Y. et al. Sci. Adv. 6, eabb5419 (2020): https://doi.org/10.1126/sciadv.abb5419
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Schöppe, J., Ehrenmann, J., Waltenspühl, Y. et al. Universal platform for the generation of thermostabilized GPCRs that crystallize in LCP. Nat Protoc 17, 698–726 (2022). https://doi.org/10.1038/s41596-021-00660-9
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DOI: https://doi.org/10.1038/s41596-021-00660-9
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