The Effect of a Widespread Cancer-Causing Mutation on the Inactive to Active Dynamics of the B-Raf Kinase

Abstract

Protein kinases play a key role in regulating cellular processes. Kinase dysfunction can lead to disease, making them an attractive target for drug design. The B-Raf kinase is a key target for the treatment of melanoma since a single mutation (V600E) is found in more than 50% of all malignant melanomas. Despite the importance of B-Raf in melanoma treatment, the molecular mechanism by which the mutation increases kinase activity remains elusive. Since kinases are tightly regulated by a conformational transition between an active and inactive state, which is difficult to capture experimentally, large-scale enhanced-sampling simulations are performed to examine the mechanism by which the V600E mutation enhances the activity of the B-Raf monomer. The results reveal that the mutation has a twofold effect. First, the mutation increases the barrier of the active to inactive transition trapping B-Raf in the active state. The mutation also increases the flexibility of the activation loop which might speed-up the rate-limiting step of phosphorylation. Both effects can be explained by the formation of salt-bridges with the Glu600 residue.

Exploring Molecular Mechanisms of Paradoxical Activation in the BRAF Kinase Dimers: Atomistic Simulations of Conformational Dynamics and Modeling of Allosteric Communication Networks and Signaling Pathways

“…MD simulations and binding free energy calculations have been employed in computational studies of type II DFG-out inhibitors in complexes with the BRAF-V600E mutant [ 75 ]. Enhanced MD simulations have suggested a mechanism in which oncogenic BRAF mutations could kinetically trap the kinase in the active form by increasing the barriers of conformational transitions between the inactive and active kinase states [ 76 ]. By combining MD simulations and protein structure network analysis, we have recently examined binding of BRAF dimers with several type I and type II inhibitors [ 77 ].…”

Phosphorylation of RAF Kinase Dimers Drives Conformational Changes that Facilitate Transactivation

“…Intriguingly, these salt bridges are primarily interprotomer salt bridges, which are formed between the NtA motif and positive residues located either upstream of the NtA motif or at the C‐terminal end of the αC‐helix, the orientation of which plays an important role in kinase activation 12a, 13…”

Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

“…Furthermore, we have also addressed a major obstacle of non-disruptive labeling of target regions that are key for the activation process, and therefore functionally sensitive, by applying the genetically encoded, site-specific incorporation of unnatural amino acids (UAAs), coupled with rapid and highly specific labeling with a chemical reporter33343536373839. Importantly, using this novel type of FRET-based construct, combined with MD simulations, we show that FGFR1 KD displays switch-like activation owing to a high free energy barrier between its non-phosphorylated and phosphorylated states, contrasting some recent examples for other kinases supporting preexisting equilibrium242640.…”

“…Due to the difficulties in obtaining experimental data, molecular dynamics (MD) simulations have been performed more extensively, providing theoretical calculations and working models for dynamic behavior of kinases212223242526272829. These studies have also highlighted the need to use a wider range of approaches to illuminate the atomistic dynamics that underpin activation mechanisms or drug binding.…”