Systems-level effects of allosteric perturbations to a model molecular switch

Molecular switch proteins whose cycling between states is controlled by opposing regulators 1 , 2 are central to biological signal transduction. As switch proteins function within highly connected interaction networks 3 , the fundamental question arises of how functional specificity is achieved when...

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Published in:	Nature (London) Vol. 599; no. 7883; pp. 152 - 157
Main Authors:	Perica, Tina, Mathy, Christopher J. P., Xu, Jiewei, Jang, Gwendolyn Μ., Zhang, Yang, Kaake, Robyn, Ollikainen, Noah, Braberg, Hannes, Swaney, Danielle L., Lambright, David G., Kelly, Mark J. S., Krogan, Nevan J., Kortemme, Tanja
Format:	Journal Article
Language:	English
Published:	London Nature Publishing Group UK 04-11-2021 Nature Publishing Group
Subjects:	38/47 38/70 631/1647/2067 631/45/612/1232 631/553/2711 631/57/2272/2273 631/92/360 82/58 82/6 82/80 82/83 Allosteric properties Allosteric Regulation - genetics Binding sites Binding Sites - genetics Biological activity Catalytic Domain - genetics Cell cycle Genes GTPase-Activating Proteins - metabolism Guanine Nucleotide Exchange Factors - metabolism Guanosine triphosphatases Guanosine Triphosphate - metabolism Homology Humanities and Social Sciences Interfaces Kinases Kinetics Molecular machines Monomeric GTP-Binding Proteins - genetics Monomeric GTP-Binding Proteins - metabolism multidisciplinary Mutation Nuclear Proteins - genetics Nuclear Proteins - metabolism Perturbation Point Mutation Post-translation Protein Binding - genetics Proteins Quantitative genetics Regulation Saccharomyces cerevisiae - enzymology Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae Proteins - genetics Saccharomyces cerevisiae Proteins - metabolism Science Science (multidisciplinary) Signal transduction Switches
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Summary:	Molecular switch proteins whose cycling between states is controlled by opposing regulators 1 , 2 are central to biological signal transduction. As switch proteins function within highly connected interaction networks 3 , the fundamental question arises of how functional specificity is achieved when different processes share common regulators. Here we show that functional specificity of the small GTPase switch protein Gsp1 in Saccharomyces cerevisiae (the homologue of the human protein RAN) 4 is linked to differential sensitivity of biological processes to different kinetics of the Gsp1 (RAN) switch cycle. We make 55 targeted point mutations to individual protein interaction interfaces of Gsp1 (RAN) and show through quantitative genetic 5 and physical interaction mapping that Gsp1 (RAN) interface perturbations have widespread cellular consequences. Contrary to expectation, the cellular effects of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle and not by the targeted interfaces. Instead, we show that interface mutations allosterically tune the GTPase cycle kinetics. These results suggest a model in which protein partner binding, or post-translational modifications at distal sites, could act as allosteric regulators of GTPase switching. Similar mechanisms may underlie regulation by other GTPases, and other biological switches. Furthermore, our integrative platform to determine the quantitative consequences of molecular perturbations may help to explain the effects of disease mutations that target central molecular switches. Interface mutations in the GTPase switch protein Gsp1 (the yeast homologue of human RAN) allosterically affect the kinetics of the switch cycle, revealing a systems-level mechanism of multi-specificity.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Author Contributions. T.P., C.J.P.M., N.J.K. and T.K. identified and developed the core questions. T.P. and C.J.P.M. performed the bulk of the experiments and data analysis. J.X. and T.P. performed the E-MAP screens. G.M.J. performed the pull-down experiments. D.L.S. and R.K. performed the MS experiments and together with T.P. analyzed the data. N.O. contributed to design of Gsp1 mutants. H.B. contributed to E-MAP analysis. M.J.S.K. suggested the NMR studies. C.J.P.M. and M.J.S.K. performed the NMR experiments and analyzed the data. T.P. performed the kinetics experiments. D.G.L. contributed to the analysis of the kinetics data. T.P., C.J.P.M, and Y.Z. purified the proteins. Y.Z. performed the Western blot experiments. T.P., C.J.P.M. and T.K. wrote the manuscript with contributions from the other authors. N.J.K. and T.K. oversaw the project.
ISSN:	0028-0836 1476-4687
DOI:	10.1038/s41586-021-03982-6