Molecular Dynamics Investigation of Oncogenic BRAF Mutations

Abstract

BRAF is one member of the RAF serine/threonine kinase family and a key component of the RAS‐RAF‐MEK‐ERK (MAPK) signaling pathway, controlling cell growth, proliferation, differentiation, migration and survival. BRAF is the most frequently mutated kinase in human cancers, with over 200 mutations discovered, among which V600E and G469A are the most common BRAF mutations. Current therapies with ATP‐competitive inhibitors are potent against BRAFV600E, yet they display “paradoxical activation” and drug resistance toward BRAFG469Aandother non‐V600 BRAF mutants. Thus, novel BRAF therapies towards non‐V600 variants are urgently needed. However, the biochemical properties of non‐V600 BRAF mutants and the cause of drug response are unclear due to the lack of structures of BRAF mutants. In this study, we applied molecular dynamics (MD) simulations to investigate the structure, dynamics and functions of oncogenic BRAFG469A, in comparison with BRAFWT and BRAFV600E. For each BRAF type (WT or mutant), MD simulations are carried out for its monomer and dimer with or without 1 or 2 bound vemurafenib in explicit aqueous solution. Our work provides atomic information on mechanism of aberrant activation by G469A mutation, “paradoxical activation” and structural dynamics of the nucleotide pocket, activation loop, and dimer and inhibitor/BRAF interfaces. G469A mutation makes hydrophobic contact between methyl of A469 with alkyl chain of K483 in the binding pocket. This stabilizes the K483‐E501 salt‐bridge, which functions to bring αC‐helix towards the active site. Thus, G469A stabilizes the active conformation by positioning αC‐helix close to the P‐loop through increasingly stabilized salt‐bridge relative to WT and V600E. Consequently, our simulations illustrate inability of vemurafenib to disrupt active conformation of BRAFG469A by separating αC‐helix from the nucleotide binding pocket. This decreased structural fluctuation upon inhibitor binding to one protomer explains the ability of “paradoxical activation” of BRAF dimer through allosteric activation.