The Mechanism of Activation of Monomeric B-Raf V600E

Abstract

Oncogenic mutations in the serine/threonine kinase B-Raf, particularly the V600E mutation, are frequent in cancer, making it a major drug target. Although much is known about B-Raf's active and inactive states, questions remain about the mechanism by which the protein changes between these two states. Here, we utilize molecular dynamics to investigate both wild-type and V600E B-Raf to gain mechanistic insights into the impact of the Val to Glu mutation. The results show that the wild-type and mutant follow similar activation pathways involving an extension of the activation loop and an inward motion of the αC-helix. The V600E mutation, however, destabilizes the inactive state by disrupting hydrophobic interactions present in the wild-type structure while the active state is stabilized through the formation of a salt bridge between Glu600 and Lys507. Additionally, when the activation loop is extended, the αC-helix is able to move between an inward and outward orientation as long as the DFG motif adopts a specific orientation. In that orientation Phe595 rotates away from the αC-helix, allowing the formation of a salt bridge between Lys483 and Glu501. These mechanistic insights have implications for the development of new Raf inhibitors.

Mechanism of activation and the rewired network: New drug design concepts

“…As an example, B‐Raf mutations have been grouped into three classes 241 (Figure 3). B‐Raf V600E mutations belong to Class I. Mutations falling into Class I activate Raf by mimicking activation loop phosphorylation, causing B‐Raf to adopt an active configuration 280–282 . Class II includes constitutive dimers.…”