The structural basis of allosteric communication networks of Ras GTPases and their oncogenic mutants

Abstract

The Ras superfamily of small GTPases are centrally involved critical cellular processes, including cell proliferation, differentiation, and survival. Ras proteins function through a molecular switch mechanism, and in the active GTP-bound state can signal to downstream partners, regulated by GEFs. Deactivation is achieved through GAP activation allowing for hydrolysis of GTP, and disruption to hydrolysis leads to overstimulation of pathways such as Ras/Raf/MEK/ERK which causes cancer. H-Ras features a distant allosteric site that interacts with GTP at the active site via a water-mediated hydrogen bonding network. Discovery of this site has led to the investigation of the role of intrinsic hydrolysis promoted by Raf. In its activated form, Ras samples conformational states which differ among the isoforms, and is influenced by point mutations, leading to varying outcomes despite their ~85% sequence identity. Initially, it was assumed that H-Ras was a suitable model for the other isoforms, and thus is represented prominently in the literature. However, K-Ras is more frequently mutated, and we have discovered differences in the highly conserved active sites of the isoforms. To gain a greater understanding of the mechanisms driving these differences, MD simulations were compared to experimental data, and analyzed for accessibility to specific conformational states. We also apply MD simulations to K-Ras oncogenic mutations which have been shown to favor specific conformations that are hypothesized promote overactivation of downstream pathways. To gain a greater understanding of K-Ras compared to H-Ras, we obtained 1NMR resonance peaks associated with sensors of the active site. To assess the conformational states, we designed a mutant that more readily accesses the catalytically competent state of Ras, as opposed to other mutations which more readily access more disordered conformation within state 2. From there, exploration of the protonation state of GTP in the Ras mechanism and the insights of the conformational states in the isoforms are tested in superfamily member Arf1. These efforts can possibly inform drug discovery targeting efforts about unique states accessed by mutations of K-Ras and other Ras superfamily proteins.