A complete allosteric map of a GTPase switch in its native cellular network

Christopher J P Mathy; Parul Mishra; Julia M Flynn; Tina Perica; David Mavor; Daniel N A Bolon; Tanja Kortemme

doi:10.1016/j.cels.2023.01.003

A complete allosteric map of a GTPase switch in its native cellular network

Cell Syst. 2023 Mar 15;14(3):237-246.e7. doi: 10.1016/j.cels.2023.01.003. Epub 2023 Feb 17.

Authors

Christopher J P Mathy¹, Parul Mishra², Julia M Flynn³, Tina Perica⁴, David Mavor³, Daniel N A Bolon⁵, Tanja Kortemme⁶

Affiliations

¹ Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94158, USA; The UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, San Francisco, San Francisco, CA 94158, USA.
² Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, MA 01605, USA; School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India.
³ Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
⁴ Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94158, USA.
⁵ Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address: dan.bolon@umassmed.edu.
⁶ Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94158, USA; The UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, San Francisco, San Francisco, CA 94158, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA. Electronic address: tanjakortemme@gmail.com.

Abstract

Allosteric regulation is central to protein function in cellular networks. A fundamental open question is whether cellular regulation of allosteric proteins occurs only at a few defined positions or at many sites distributed throughout the structure. Here, we probe the regulation of GTPases-protein switches that control signaling through regulated conformational cycling-at residue-level resolution by deep mutagenesis in the native biological network. For the GTPase Gsp1/Ran, we find that 28% of the 4,315 assayed mutations show pronounced gain-of-function responses. Twenty of the sixty positions enriched for gain-of-function mutations are outside the canonical GTPase active site switch regions. Kinetic analysis shows that these distal sites are allosterically coupled to the active site. We conclude that the GTPase switch mechanism is broadly sensitive to cellular allosteric regulation. Our systematic discovery of new regulatory sites provides a functional map to interrogate and target GTPases controlling many essential biological processes.

Keywords: GTPases; Gsp1; Ran; allostery; cellular regulation; mutational scanning; protein networks.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Allosteric Regulation / genetics
Allosteric Site
GTP Phosphohydrolases* / genetics
Kinetics
Proteins*

Substances

GTP Phosphohydrolases
Proteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding