Deconstruction of the Ras switching cycle through saturation mutagenesis
- University of California, Berkeley, United States
- Ragon Institute of MGH, MIT and Harvard, United States
- University of California, San Francisco, United States
- University of Texas Southwestern Medical Center, United States
Abstract
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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Author details
Funding
Howard Hughes Medical Institute
- Pradeep Bandaru
- Neel H Shah
- Moitrayee Bhattacharyya
- Yasushi Kondo
- Joshua C Cofsky
- Christine L Gee
National Institutes of Health
- John P Barton
- Arup K Chakraborty
Damon Runyon Cancer Research Foundation
- Neel H Shah
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2017, Bandaru et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Deconstruction of the Ras switching cycle through saturation mutagenesis
eLife 6:e27810.
https://doi.org/10.7554/eLife.27810
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