Deconstruction of the ras switching cycle through saturation mutagenesis

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

Research output: Contribution to journalArticlepeer-review

55 Scopus citations


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.

Original languageEnglish (US)
Article numbere27810
StatePublished - Jul 7 2017

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)


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