Biochemical Characterization of Full‐Length Oncogenic BRAF^V600E together with Molecular Dynamics Simulations Provide Insight into the Activation and Inhibition Mechanisms of RAF Kinases

Abstract

The most prevalent BRAF mutation, V600E, occurs frequently in melanoma and other cancers. Although extensive progress has been made toward understanding the biology of RAF kinases, little in vitro characterization of full‐length BRAFV600E is available. Herein, we show the successful purification of active, full‐length BRAFV600E from mammalian cells for in vitro experiments. Our biochemical characterization of intact BRAFV600E together with molecular dynamics (MD) simulations of the BRAF kinase domain and cell‐based assays demonstrate that BRAFV600E has several unique features that contribute to its tumorigenesis. Firstly, steady‐state kinetic analyses reveal that purified BRAFV600E is more active than fully activated wild‐type BRAF; this is consistent with the notion that elevated signaling output is necessary for transformation. Secondly, BRAFV600E has a higher potential to form oligomers, despite the fact that the V600E substitution confers constitutive kinase activation independent of an intact side‐to‐side dimer interface. Thirdly, BRAFV600E bypasses inhibitory P‐loop phosphorylation to enforce the necessary elevated signaling output for tumorigenesis. Together, these results provide new insight into the biochemical properties of BRAFV600E, complementing the understanding of BRAF regulation under normal and disease conditions.

The Mechanism of Activation of Monomeric B-Raf V600E

“…Also presented in Figure S7b is the center of mass distance between the αC-helix in the N-lobe and αE-helix in the C-lobe. [85] These data follow the same trends as shown in Figure 3 and show that the formation of the salt bridge between Lys483 and Glu501 is tied to the inward motion of the αC-helix.…”

The mechanism of activation of monomeric B-Raf V600E

“…The simulations that begin with intermediate or outward oriented αC-helix display distinct behavior from their wild-type counterparts. Although none of these simulations moved from the inactive to the active state, as other simulations have reported [85] , there are indications how this mutation may destabilize the inactive configuration. Fig.…”

“…This work builds upon previous simulation studies of monomeric B-Raf to further our understanding of the mechanism of activation for B-Raf V600E. Previous MD simulations of monomeric B-Raf with an empty ATP binding pocket have found that wild-type B-Raf with an initially active configuration will quickly convert to an inactive conformation through the formation of the AS-H1 helix and an outward movement of the αC-helix [85] . We do not observe this in our simulations which include ATP and the magnesium ion, a requirement for a kinase action.…”

“…This interaction could stabilize the residues around the DFG motif, preventing the formation of the inhibitory helix. It has also been reported that active V600E will maintain an active conformation with an inward αC-helix [85] . While most of our simulations agree with this, there were results that indicate that the αC-helix of active B-Raf V600E can switch between the inward and outward conformations yet maintain an active BLAminus XDFG orientation.…”

“…Also presented in Fig. S7 b is the center of mass distance between the αC-helix in the N-lobe and αE-helix in the C-lobe [85] . These data follow the same trends as shown in Fig.…”