Catalytically Competent Non-transforming H-RASG12P Mutant Provides Insight into Molecular Switch Function and GAP-independent GTPase Activity of RAS

Abstract

RAS mutants have been extensively studied as they are associated with development of cancer; however, H-RAS G12P mutant has remained untouched since it does not lead to transformation in the cell. To the best of our knowledge, this is the first study where structural/dynamical properties of H-RAS G12P have been investigated -in comparison to H-RAS WT , H-RAS G12D , RAF-RBD-bound and GAP-bound H-RAS WT - using molecular dynamics simulations (total of 9 μ s). We observed remarkable differences in dynamics of Y32. Specifically, it is located far from the nucleotide binding pocket in the catalytically-active GAP-bound H-RAS WT , whereas it makes close interaction with the nucleotide in signaling-active systems (H-RAS G12D , KRAS4B G12D , RAF-RBD-bound H-RAS WT ) and H-RAS WT . The accessibility of Y32 in wild type protein is achieved upon GAP binding. Interestingly; however, it is intrinsically accessible in H-RAS G12P . Considering the fact that incomplete opening of Y32 is associated with cancer, we propose that Y32 can be targeted by means of small therapeutics that can displace it from the nucleotide binding site, thus introducing intrinsic GTPase activity to RAS mutants, which cannot bind to GAP. Therefore, mimicking properties of H-RAS G12P in RAS-centered drug discovery studies has the potential of improving success rates since it acts as a molecular switch per se .

Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS

“…Herein, it is important to mention that exposed conformation of Y32 was not observed in the trajectories pertaining to RAF-RBD-bound HRAS WT as shown in our earlier study ( Ilter and Sensoy, 2019 ). Therefore, this finding suggests that exposure of Y32 might occlude the interaction interface formed between RAS and RAF-RBD.…”

“…The most probable conformational state of the binding pocket pertaining to HRAS G12D was determined by using following reaction coordinates: distance measured between (i) side-chain oxygen of residue T35 and Pγ atom of GTP, (ii) backbone amide of the residue G60 and Pγ atom of GTP, which were used to point out different conformational states of the nucleotide-bindng pocket in our previous study ( Ilter and Sensoy, 2019 ). The frames, which represent different conformational states of the nucleotide-binding pocket with respect to the above-mentioned coordinates, were selected.…”

Inhibition of mutant RAS-RAF interaction by mimicking structural and dynamic properties of phosphorylated RAS

“…Herein, it is important to mention that exposed conformation of Y32 was not observed in the trajectories pertaining to RAF-RBD-bound HRAS WT as shown in our earlier study Ilter and Sensoy (2019 ). Therefore, this finding suggests that exposure of Y32 might occlude the interaction interface formed between RAS and RAF-RBD and exposure of Y32 might facilitate water attacks to P γ of GTP, hence increasing intrinsic GTPase activity, in accordance with experimental data Bunda et al (2014 ).…”

“…In this study, we set out to investigate the impact of phosphorylation on the structure and dynamics of HRAS WT by performing atomistic MD simulations. Comparison of the trajectory pertaining to the phosphorylated RAS with previously obtained trajectories of GTP-bound HRAS WT and HRAS G12D Ilter and Sensoy (2019 ) showed that phosphorylation of Y32 increased the flexibility of both RAF-RBD and RAF-CRD (cysteine-rich domain) interfaces and pushed Switch I, in particular Y32, out of the nucleotide-binding pocket. Considering the fact that, exposed Y32 precluded RAF binding, we searched for molecules that could evoke similar rearrangements in HRAS G12D .…”