Nucleotide-dependent Structural Selection Governs c-Src Phosphorylation of Oncogenic KRas4B-G12D

Abstract

c-Src, the first identified oncogene, and KRas, one of the most frequently mutated proteins in human cancers, can activate each other to promote tumorigenesis. Although normal KRas proteins cycle between the inactive GDP bound and the active GTP bound forms, oncogenic mutants are predominantly GTP bound. Importantly, c-Src preferentially recognizes and phosphorylates the GTP-bound form of KRas but the molecular mechanism underlying this specific recognition remains unknown. Here, we employ molecular simulation tools to identify the mechanism underlying the c-Src recognition of the GTP-loaded state of the G12D mutant of KRas4B (the most prevalent in humans). Combining extensive all-atom Molecular Dynamics simulations and Markov State Models analysis, we found that the most populated states of GTP-bound KRas maintain more open and dynamic switch regions, facilitating easier access of c-Src to the phosphorylation sites of KRas (Tyr32 and Tyr64). These states are sparsely populated for GDP loaded KRas. Docking calculations refined by molecular dynamics identify two c-Src specific regions (residues 340-359 and 453-473) able to stabilize phosphorylation-competent KRas conformations. Therefore, c-Src engages highly populated macrostates of GTP-loaded KRas, while interactions with the GDP form are limited to rare conformations. These KRas conformations selectively recognized by c-Src constitute privileged targets for the rational design of peptide-based or small-molecule inhibitors that specifically target active KRas4B-G12D while sparing the inactive GDP-bound form.