Deconstruction of the Ras switching cycle through saturation mutagenesis

Bandaru, Pradeep; Shah, Neel H.; Bhattacharyya, Moitrayee; Barton, John P.; Kondo, Yasushi; Cofsky, Joshua C.; Gee, Christine L.; Chakraborty, Arup K.; Kortemme, Tanja; Ranganathan, Rama; Kuriyan, John

doi:10.7554/elife.27810

Title: Deconstruction of the Ras switching cycle through saturation mutagenesis

Journal Article · 07 July 2017 · eLife

DOI: https://doi.org/10.7554/elife.27810 · OSTI ID:1628873

^[1];

^[1]; Bhattacharyya, Moitrayee ^[1];

^[2]; Kondo, Yasushi ^[1]; Cofsky, Joshua C. ^[1];

^[1]; Chakraborty, Arup K. ^[3]; Kortemme, Tanja ^[4]; Ranganathan, Rama ^[5];

^[6]

Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). California Inst. for Quantitative Biosciences; Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.
Massachusetts Inst. of Technology (MIT) and Harvard Univ., Cambridge, MA (United States). Ragon Inst. of MGH; Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Chemical Engineering; Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Physics; Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Inst. for Medical Engineering and Science
Massachusetts Inst. of Technology (MIT) and Harvard Univ., Cambridge, MA (United States). Ragon Inst. of MGH; Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Chemical Engineering; Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Physics; Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Inst. for Medical Engineering and Science; Dept. of Chemistry; Dept. of Biological Engineering
Univ. of California, San Francisco, CA (United States). California Inst. for Quantitative Biomedical Research. Dept. of Bioengineering and Therapeutic Sciences
Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Pharmacology; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Green Center for Systems Biology; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biophysics
Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). California Inst. for Quantitative Biosciences; Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division

Ras proteins are highly conserved signaling molecules that exhibit regulated, nucleotide-dependent switching between active and inactive states. The high conservation of Ras requires mechanistic explanation, especially given the general mutational tolerance of proteins. Here, we use deep mutational scanning, biochemical analysis and molecular simulations to understand constraints on Ras sequence. Ras exhibits global sensitivity to mutation when regulated by a GTPase activating protein and a nucleotide exchange factor. Removing the regulators shifts the distribution of mutational effects to be largely neutral, and reveals hotspots of activating mutations in residues that restrain Ras dynamics and promote the inactive state. Evolutionary analysis, combined with structural and mutational data, argue that Ras has co-evolved with its regulators in the vertebrate lineage. Overall, our results show that sequence conservation in Ras depends strongly on the biochemical network in which it operates, providing a framework for understanding the origin of global selection pressures on proteins.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1628873

Journal Information:: eLife, Vol. 6; ISSN 2050-084X

Publisher:: eLife Sciences Publications, Ltd.Copyright Statement

Country of Publication:: United States

Language:: English

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