Deconstruction of the Ras switching cycle through saturation mutagenesis

• Published in: eLife Vol. 6
• Main Authors: 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
• Format: Journal Article
• Language: English
• Published: England eLife Science Publications, Ltd 07-07-2017; eLife Sciences Publications Ltd; eLife Sciences Publications, Ltd
• Subjects: allostery; Antibiotics; BASIC BIOLOGICAL SCIENCES; Biochemical analysis; Bioengineering; Biophysics; Biophysics and Structural Biology; Conserved Sequence; DNA Mutational Analysis; Drug resistance; evolution; Evolution, Molecular; Experiments; Growth rate; Guanosine triphosphatases; Humans; Life Sciences & Biomedicine - Other Topics; Medical research; Methods; Mutagenesis; Mutation; Observations; Phylogenetics; Physiological aspects; Plasmids; protein dynamics; Protein Interaction Maps; Protein structure prediction; Proteins; Ras genes; Ras protein; ras Proteins - genetics; ras Proteins - metabolism; Saturation mutagenesis; Structure; systems biology; United States > US; California; systems biology; protein dynamics; structural biology; evolution; biophysics; E. coli; S. cerevisiae; allostery
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.27810

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.