Deconstruction of the Ras switching cycle through saturation mutagenesis.

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

doi:10.7554/elife.27810

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.

Highlights

Protein structures are remarkably robust to changes in primary sequence
Most mutations shift to being near-neutral and a set of gain-of-function mutations at hotspot residues appears, where almost any change to the wild-type residue results in increased activity. We show that these hotspots of activating mutations, which form a spatially contiguous network distributed around the GTPase domain, correspond to residues that reduce spontaneous switching through allosteric coupling to the GTP binding site
The binding of Ras.GTP to Raf-Ras-binding domain (RBD) leads to transcription of an antibiotic resistance gene coding for chloramphenicol acetyltransferase (CAT)

Summary

Introduction

Protein structures are remarkably robust to changes in primary sequence. This concept dates back to the early 1960s, when Max Perutz and John Kendrew observed striking similarities between the three-dimensional structures of myoglobin and hemoglobin, despite limited sequence identity (Kendrew et al, 1954; Perutz et al, 1960; Lesk and Chothia, 1980). Studies using deep sequencing have reinforced the concept that proteins are remarkably tolerant of mutation while retaining the ability to fold and function (Bershtein et al, 2006; Roscoe et al, 2013; Fowler and Fields, 2014; Tripathi and Varadarajan, 2014; Podgornaia and Laub, 2015). ‘two-hybrid’ selection systems, combined with deep sequencing, have been utilized to determine the effects of mutations on the binding affinities of interacting proteins (Dove et al, 1997; Joung et al, 2000). This strategy has been used to probe the sensitivity to mutation of a PDZ domain-peptide interaction (McLaughlin et al, 2012). Residues in the PDZ domain are highly tolerant of mutation, except for those that govern folding, stability, and energetic interactions with the peptide target

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Results

Conclusion