The GFP thermal shift assay for screening ligand and lipid interactions to solute carrier transporters

Yurie Chatzikyriakidou, Do-Hwan Ahn, Emmanuel Nji, David Drew

Published: 2021-10-26 DOI: 10.1038/s41596-021-00619-w

high-throughput screening

GFP thermal shift assay

SLC transporters

ligand interactions

lipid–protein interactions

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Solute carrier (SLC) transporters represent the second-largest fraction of the membrane proteome after G-protein-coupled receptors, but have been underutilized as drug targets and the function of many members of this family is still unknown. They are tech
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ACKNOWLEDGMENTS

Cesar-Razquin, A. et al. A call for systematic research on solute carriers. Cell 162, 478–487 (2015).
Lin, L., Yee, S. W., Kim, R. B. & Giacomini, K. M. SLC transporters as therapeutic targets: emerging opportunities. Nat. Rev. Drug Discov. 14, 543–560 (2015).
Drew, D. & Boudker, O. Shared molecular mechanisms of membrane transporters. Annu. Rev. Biochem. 85, 543–572 (2016).
Hediger, M. A. et al. The ABCs of solute carriers: physiological, pathological and therapeutic implications of human membrane transport proteinsIntroduction. Pflug. Arch. 447, 465–468 (2004).
Superti-Furga, G. et al. The RESOLUTE consortium: unlocking SLC transporters for drug discovery. Nat. Rev. Drug Discov. 19, 429–430 (2020).
Colas, C., Ung, P. M. & Schlessinger, A. SLC transporters: structure, function, and drug discovery. MedChemComm 7, 1069–1081 (2016).
Scheen, A. J. Sodium-glucose cotransporter type 2 inhibitors for the treatment of type 2 diabetes mellitus. Nat. Rev. Endocrinol. 16, 556–577 (2020).
Yu, H. B., Li, M., Wang, W. P. & Wang, X. L. High throughput screening technologies for ion channels. Acta Pharmacol. Sin. 37, 34–43 (2016).
Yasi, E. A., Kruyer, N. S. & Peralta-Yahya, P. Advances in G protein-coupled receptor high-throughput screening. Curr. Opin. Biotechnol. 64, 210–217 (2020).
Scalise, M., Pochini, L., Giangregorio, N., Tonazzi, A. & Indiveri, C. Proteoliposomes as tool for assaying membrane transporter functions and interactions with xenobiotics. Pharmaceutics 5, 472–497 (2013).
Majd, H. et al. Screening of candidate substrates and coupling ions of transporters by thermostability shift assays. eLife http://doi.org/10.7554/eLife.38821 (2018).
Volpe, D. A. Transporter assays as useful in vitro tools in drug discovery and development. Expert Opin. Drug Discov. 11, 91–103 (2016).
Sonoda, Y. et al. Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures. Structure 19, 17–25 (2011).
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).
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).
Bazzone, A., Barthmes, M. & Fendler, K. SSM-based electrophysiology for transporter research. Methods Enzymol. 594, 31–83 (2017).
Amati, A. M., Graf, S., Deutschmann, S., Dolder, N. & von Ballmoos, C. Current problems and future avenues in proteoliposome research. Biochem. Soc. Trans. 48, 1473–1492 (2020).
Martinez Molina, D. & Nordlund, P. The cellular thermal shift assay: a novel biophysical assay for in situ drug target engagement and mechanistic biomarker studies. Annu. Rev. Pharmacol. Toxicol. 56, 141–161 (2016).
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).
Lo, M. C. et al. Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Anal. Biochem. 332, 153–159 (2004).
Newstead, S. et al. Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2. EMBO J. 30, 417–426 (2011).
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).
Tomasiak, T. M. et al. General qPCR and plate reader methods for rapid optimization of membrane protein purification and crystallization using thermostability assays. Curr. Protoc. Protein Sci. 77, 29 11 21–14 (2014).
Allison, T. M. et al. Quantifying the stabilizing effects of protein-ligand interactions in the gas phase. Nat. Commun. 6, 8551 (2015).
Crichton, P. G. et al. Trends in thermostability provide information on the nature of substrate, inhibitor, and lipid interactions with mitochondrial carriers. J. Biol. Chem. 290, 8206–8217 (2015).
Oates, J. et al. The role of cholesterol on the activity and stability of neurotensin receptor 1. Biochim. Biophys. Acta 1818, 2228–2233 (2012).
Nji, E., Chatzikyriakidou, Y., Landreh, M. & Drew, D. An engineered thermal-shift screen reveals specific lipid preferences of eukaryotic and prokaryotic membrane proteins. Nat. Commun. 9, 4253 (2018).
Nji, E., Gulati, A., Qureshi, A. A., Coincon, M. & Drew, D. Structural basis for the delivery of activated sialic acid into Golgi for sialyation. Nat. Struct. Mol. Biol. 26, 415–423 (2019).
Almqvist, H. et al. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil. Nat. Commun. 7, 11040 (2016).
Savitski, M. M. et al. Tracking cancer drugs in living cells by thermal profiling of the proteome. Science 346, 1255784 (2014).
Reinhard, F. B. et al. Thermal proteome profiling monitors ligand interactions with cellular membrane proteins. Nat. Methods 12, 1129–1131 (2015).
Carnero Corrales, M. A. et al. Thermal proteome profiling identifies the membrane-bound purinergic receptor P2X4 as a target of the autophagy inhibitor indophagolin. Cell Chem. Biol. http://doi.org/10.1016/j.chembiol.2021.02.017 (2021).
Hashimoto, M., Girardi, E., Eichner, R. & Superti-Furga, G. Detection of chemical engagement of solute carrier proteins by a cellular thermal shift assay. ACS Chem. Biol. 13, 1480–1486 (2018).
Martens, C. et al. Direct protein-lipid interactions shape the conformational landscape of secondary transporters. Nat. Commun. 9, 4151 (2018).
Bolla, J. R., Agasid, M. T., Mehmood, S. & Robinson, C. V. Membrane protein-lipid interactions probed using mass spectrometry. Annu. Rev. Biochem. 88, 85–111 (2019).
van ‘t Klooster, J. S. et al. Membrane lipid requirements of the lysine transporter Lyp1 from Saccharomyces cerevisiae. J. Mol. Biol. 432, 4023–4031 (2020).
Scanlon, S. M., Williams, D. C. & Schloss, P. Membrane cholesterol modulates serotonin transporter activity. Biochemistry 40, 10507–10513 (2001).
Barrera, N. P., Zhou, M. & Robinson, C. V. The role of lipids in defining membrane protein interactions: insights from mass spectrometry. Trends Cell Biol. 23, 1–8 (2013).
Bechara, C. & Robinson, C. V. Different modes of lipid binding to membrane proteins probed by mass spectrometry. J. Am. Chem. Soc. 137, 5240–5247 (2015).
Cong, X. et al. Determining membrane protein-lipid binding thermodynamics using native mass spectrometry. J. Am. Chem. Soc. 138, 4346–4349 (2016).
Cong, X., Liu, Y., Liu, W., Liang, X. & Laganowsky, A. Allosteric modulation of protein-protein interactions by individual lipid binding events. Nat. Commun. 8, 2203 (2017).
Gault, J. et al. High-resolution mass spectrometry of small molecules bound to membrane proteins. Nat. Methods 13, 333–336 (2016).
Winkelmann, I. et al. Structure and elevator mechanism of the mammalian sodium/proton exchanger NHE9. EMBO J. http://doi.org/10.15252/embj.2020105908 (2020).
Kawate, T. & Gouaux, E. Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14, 673–681 (2006).
Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).
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).
Hu, N. J. et al. GFP-based expression screening of membrane proteins in insect cells using the baculovirus system. Methods Mol. Biol. 1261, 197–209 (2015).
Vedadi, M., Arrowsmith, C. H., Allali-Hassani, A., Senisterra, G. & Wasney, G. A. Biophysical characterization of recombinant proteins: a key to higher structural genomics success. J. Struct. Biol. 172, 107–119 (2010).
Bantscheff, M., Scholten, A. & Heck, A. J. Revealing promiscuous drug-target interactions by chemical proteomics. Drug Discov. Today 14, 1021–1029 (2009).
Kang, H. J., Lee, C. & Drew, D. Breaking the barriers in membrane protein crystallography. Int. J. Biochem. Cell Biol. 45, 636–644 (2013).
Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4, 706–731 (2009).
Mattle, D. et al. Ligand channel in pharmacologically stabilized rhodopsin. Proc. Natl Acad. Sci. USA 115, 3640–3645 (2018).
Landreh, M. et al. Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters. Nat. Commun. 8, 13993 (2017).
Gupta, K. et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature 541, 421–424 (2017).
Bird, L. E. et al. Expression screening of integral membrane proteins by fusion to fluorescent reporters. Adv. Exp. Med. Biol. 922, 1–11 (2016).

The GFP thermal shift assay for screening ligand and lipid interactions to solute carrier transporters

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