Identification and Characterization of a B-Raf Kinase α-Helix Critical for the Activity of MEK Kinase in MAPK Signaling

Abstract

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and ~3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-Raf V600E expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-Raf V600E . We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry, and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-Raf V600E -induced melanomas.

The mechanism of activation of monomeric B-Raf V600E

“…First, most crystal structures (except for 6U2H) had lower B -factor values around the αG-helix, particularly those crystal structures of Raf/MEK dimers. The αG-helix is important in B-Raf/MEK binding [84] , so we would expect these residues to be more flexible in monomers. Indeed, previous simulations of monomeric B-Raf with an empty ligand binding pocket have found a possible active-inactive transition state in which this helix is partially unfolded [45] .…”

The Mechanism of Activation of Monomeric B-Raf V600E

“…First, most crystal structures (except for 6U2H) had lower B -factor values around the αG-helix, particularly those crystal structures of Raf/MEK dimers. The αG-helix is important in B-Raf/MEK binding,[84] so we would expect these residues to be more flexible in monomers. Indeed, previous simulations of monomeric B-Raf with an empty ligand binding pocket have found a possible active-inactive transition state in which this helix is partially unfolded.…”

Comprehensive analysis of crystal structure, spectroscopic properties, quantum chemical insights, and molecular docking studies of two pyrazolopyridine compounds: potential anticancer agents

“…41 In contrast, the active sites of the kinases BRAF, MEK2, and PDGFRA have neutral electrostatic characteristics. [42][43][44] JAK, AKT1, and AKT2 have positively charged binding sites, making them attractive to negatively charged ligands. [45][46][47] These electrostatic properties have signicant implications for the affinities of compounds 4a and 4b.…”