B-Raf is a
serine/threonine-specific protein kinase. As such, it catalyzes the phosphorylation of
serine and
threonine residues in a
consensus sequence on target proteins by
ATP, yielding
ADP and a phosphorylated protein as products.
Activation Relieving CR1 autoinhibition The kinase (CR3) domain of human
Raf kinases is inhibited by two mechanisms:
autoinhibition by its own
regulatory Ras-
GTP-binding CR1 domain and a lack of
post-translational phosphorylation of key serine and
tyrosine residues (S338 and Y341 for c-Raf) in the CR2 hinge region. During B-Raf activation, the protein's
autoinhibitory CR1 domain first binds Ras-GTP's
effector domain to the CR1 Ras-binding domain (RBD) to release the kinase CR3 domain like other members of the human
Raf kinase family. The CR1-Ras interaction is later strengthened through the binding of the
cysteine-rich subdomain (CRD) of CR1 to Ras and
membrane phospholipids. This allows the negatively charged phosphoserine to immediately repel CR1 through steric and electrostatic interactions once the regulatory domain is unbound, freeing the CR3 kinase domain to interact with substrate proteins.
CR3 domain activation After the autoinhibitory CR1 regulatory domain is released, B-Raf's CR3
kinase domain must change to its
ATP-binding active
conformer before it can catalyze
protein phosphorylation. In the inactive conformation, F595 of the DFG motif blocks the
hydrophobic adenine binding pocket while
activation loop residues form hydrophobic interactions with the P-loop, stopping ATP from accessing its binding site. When the activation loop is phosphorylated, the negative charge of the phosphate is unstable in the hydrophobic environment of the P-loop. As a result, the activation loop changes
conformation, stretching out across the C-lobe of the
kinase domain. In this process, it forms stabilizing
β-sheet interactions with the β6 strand. Meanwhile, the phosphorylated residue approaches K507, forming a stabilizing
salt bridge to lock the activation loop into place. The DFG motif changes conformation with the activation loop, causing F595 to move out of the adenine nucleotide binding site and into a hydrophobic pocket bordered by the
αC and αE helices. Together, DFG and activation loop movement upon phosphorylation open the ATP
binding site. Since all other substrate-binding and catalytic domains are already in place, phosphorylation of the activation loop alone activates B-Raf's kinase domain through a chain reaction that essentially removes a lid from an otherwise-prepared active site.
Mechanism of catalysis To effectively catalyze protein phosphorylation via the bimolecular substitution of serine and threonine residues with
ADP as a
leaving group, B-Raf must first bind ATP and then stabilize the
transition state as the γ-phosphate of ATP is transferred.
ATP binding B-Raf binds ATP by anchoring the adenine nucleotide in a
nonpolar pocket (yellow, Figure 1) and orienting the molecule through hydrogen-bonding and electrostatic interactions with phosphate groups. In addition to the P-loop and DFG motif phosphate binding described above, K483 and E501 play key roles in stabilizing non-transferable phosphate groups. The positive charge on the primary
amine of K483 allows it to stabilize the negative charge on ATP α- and β-phosphate groups when ATP binds. When ATP is not present, the negative charge of the E501
carboxyl group balances this charge.
Phosphorylation Once ATP is bound to the B-Raf kinase domain, D576 of the catalytic loop activates a substrate hydroxyl group, increasing its nucleophilicity to kinetically drive the phosphorylation reaction while other catalytic loop residues stabilize the transition state (Figure 2). N581 chelates the divalent magnesium cation associated with ATP to help orient the molecule for optimal substitution. K578 neutralizes the negative charge on the γ-phosphate group of ATP so that the activated ser/thr substrate residue will not experience as much electron-electron repulsion when attacking the phosphate. After the phosphate group is transferred, ADP and the new phosphoprotein are released.
Inhibitors Since constitutively active B-Raf mutants commonly cause cancer (see Clinical Significance) by excessively signaling cells to grow, inhibitors of B-Raf have been developed for both the inactive and active conformations of the kinase domain as cancer therapeutic candidates.
Sorafenib ring further prohibits DFG motif and activation loop movement to the active confermer via steric blockage. BAY43-9006 (
Sorafenib, Nexavar) is a V600E
mutant B-Raf and
C-Raf inhibitor approved by the
FDA for the treatment of primary
liver and
kidney cancer. Bay43-9006 disables the B-Raf
kinase domain by locking the enzyme in its inactive form. The inhibitor accomplishes this by blocking the ATP binding pocket through high-
affinity for the kinase domain. It then binds key activation loop and
DFG motif residues to stop the movement of the activation loop and DFG motif to the active conformation. Finally, a trifluoromethyl phenyl moiety sterically blocks the DFG motif and activation loop active conformation site, making it impossible for the kinase domain to shift conformation to become active. The distal
pyridyl ring of BAY43-9006 anchors in the hydrophobic nucleotide-binding pocket of the kinase N-lobe, interacting with W531, F583, and F595. The hydrophobic interactions with catalytic loop F583 and DFG motif F595 stabilize the inactive conformation of these structures, decreasing the likelihood of enzyme activation. Further hydrophobic interaction of K483, L514, and T529 with the center phenyl ring increase the affinity of the kinase domain for the inhibitor. Hydrophobic interaction of F595 with the center ring as well decreases the energetic favorability of a DFG conformation switch further. Finally, polar interactions of BAY43-9006 with the kinase domain continue this trend of increasing enzyme affinity for the inhibitor and stabilizing DFG residues in the inactive conformation. E501 and C532 hydrogen bond the
urea and pyridyl groups of the inhibitor respectively while the
urea carbonyl accepts a hydrogen bond from D594's backbone
amide nitrogen to lock the DFG motif in place. The trifluoromethyl phenyl moiety cements the thermodynamic favorability of the inactive conformation when the kinase domain is bound to BAY43-9006 by sterically blocking the hydrophobic pocket between the αC and αE helices that the DFG motif and activation loop would inhabit upon shifting to their locations in the active conformation of the protein.
Vemurafenib PLX4032 (
Vemurafenib) is a V600
mutant B-Raf inhibitor approved by the
FDA for the treatment of late-stage
melanoma. Unlike
BAY43-9006, which inhibits the inactive form of the kinase domain, Vemurafenib inhibits the active "DFG-in" form of the kinase, firmly anchoring itself in the ATP-binding site. By inhibiting only the active form of the kinase, Vemurafenib selectively inhibits the proliferation of cells with unregulated B-Raf, normally those that cause
cancer. Since Vemurafenib only differs from its precursor, PLX4720, in a
phenyl ring added for
pharmacokinetic reasons, PLX4720's mode of action is equivalent to Vemurafenib's. PLX4720 has good affinity for the ATP binding site partially because its anchor region, a 7-aza
indole bicyclic, only differs from the natural adenine that occupies the site in two places where nitrogen atoms have been replaced by carbon. This enables strong intermolecular interactions like N7 hydrogen bonding to C532 and N1 hydrogen bonding to Q530 to be preserved. Excellent fit within the ATP-binding hydrophobic pocket (C532, W531, T529, L514, A481) increases binding affinity as well.
Ketone linker hydrogen bonding to water and difluoro-phenyl fit in a second hydrophobic pocket (A481, V482, K483, V471, I527, T529, L514, and F583) contribute to the exceptionally high binding affinity overall. Selective binding to active Raf is accomplished by the terminal propyl group that binds to a Raf-selective pocket created by a shift of the αC helix. Selectivity for the active conformation of the kinase is further increased by a pH-sensitive deprotonated
sulfonamide group that is stabilized by hydrogen bonding with the backbone peptide NH of D594 in the active state. In the inactive state, the inhibitor's sulfonamide group interacts with the backbone
carbonyl of that residue instead, creating repulsion. Thus, Vemurafenib binds preferentially to the active state of B-Raf's kinase domain. == Clinical significance ==