Evolutionary constraints on the sequence of Ras

Abstract/nEvolutionary constraints on the sequence of Ras/nby/nPradeep Bandaru/nDoctor of Philosophy in Molecular and Cellular Biology/nUniversity of California, Berkeley/nProfessor John Kuriyan, Chair/nRas proteins are highly conserved signaling molecules that exhibit regulated, nucleotide-dependent switching between an active GTP-bound state that transduces signals by binding to effector proteins, and an inactive GDP-bound state that cannot bind to effectors. The high conservation of Ras requires mechanistic explanation, especially given that proteins are generally robust to mutation, a concept that was first established from early structural and phylogenetic analysis of hemoglobin by Max Perutz and John Kendrew./nDuring my thesis research, I adapted a two-hybrid selection system to analyze how mutations affect the functional cycle of human H-Ras, with the ultimate goal of understanding the constraints on the sequence of Ras that give rise to its high evolutionary conservation. My strategy was to isolate just the minimal biochemical network that defines this cycle, comprising Ras, its effector Raf, a GTPase accelerating protein (GAP), and a guanine-nucleotide exchange factor (GEF). Using this selection system, I analyzed the sensitivity of every residue in Ras to mutation in the context of this network, while excluding the effects of the membrane and additional regulatory factors. This approach provided an opportunity, for the first time, to use deep mutational scanning approaches to study how local regulatory networks influence the mutational sensitivity and phenotypic plasticity of key signaling molecules./nI found that Ras exhibits global sensitivity to mutation when regulated by a GAP and a GEF, effectively displaying global constraints that result in the majority of mutations leading to a modest decrease in Ras function. In the absence of regulators, Ras shows considerable tolerance to mutation, as seen previously in saturation mutagenesis experiments on other proteins, where the distribution of mutational effects shifted to be largely neutral. Surprisingly, the analysis of Ras in the absence of regulators also revealed allosteric hotspots of activating mutations in residues that restrain Ras dynamics and promote the inactive, GDP-bound state. This showed that structural fold of Ras is intrinsically capable of accommodating sequence changes that, in evolution, could lead to the acquisition of new function, but could also lead to unwanted Ras activation in disease. Indeed, oncogenic mutations that disturb the switching mechanism of Ras result in aberrant signaling and cancer, highlighted by the fact that Ras is one of the most important proto-oncogenes in the human genome./nAltogether, my research shows that the local regulatory network places a stringent constraint on the sequence of Ras, and also creates the potential conditions in which it is susceptible to activating mutations. This extended previous observations that mutational sensitivity in proteins is strongly dependent on the selective conditions in which the protein operates. From a practical perspective, though small molecule inhibitors of Ras have yet to achieve clinical relevance despite a concerted effort to obtain such inhibitors, my research also provides a roadmap of allosteric hotspots of Ras activation that can be exploited to design novel cancer therapeutics.

Advisor: Kuriyan, John