B-Raf is part of a conserved signaling pathway, the Ras-Raf-MEK-ERK pathway, which relays extracellular signals to the nucleus in order to regulate cell proliferation, differentiation, and death (1-6). B-Raf is one of three serine/threonine kinases activated through phosphorylation by Ras (1-4). Raf kinases activate MEK by phosphorylation, which then phosphorylates and activates ERK (1-6). Mutations in the Ras-Raf-MEK-ERK signaling pathway are associated with human cancers (1-6,9). About 60% of malignant melanomas contain mutated B-Raf (2,3,6). Most mutations affect the B-Raf kinase domain (PDB ID: 1UWH) in Homo sapiens (1,3,4,6). 

      Raf proteins contain three regions (1,6). The first and second regions, toward the N-terminal end, are mostly regulatory (1). The B-Raf kinase domain is contained in the third region near the C-terminus of the protein (1,6). The B-Raf kinase domain includes 276 residues, encompassing B-Raf residues 447-722. It has a molecular weight of 63810.31 Da and an isoelectric point of 8.5. The B-Raf kinase region adopts a tertiary structure similar to other kinases, with a small lobe and a large lobe separated by a catalytic cleft (1,2). The secondary structure of the small lobe is predominately beta sheets, while the large lobe contains mainly alpha helices (1). B-Raf kinase domain quaternary structure is comprised of two interacting kinase domain chains. 

      Human C-Raf kinase domain (also known as Raf-1 kinase) (PDB ID: 3OMV) is another of the three Raf serine/threonine kinases (1-4). C-Raf kinase has a very similar structure to the B-Raf kinase domain (7,8). According to the PSI-BLAST server, C-Raf has an E-value of 2x10-164 for primary structure similarities when compared with B-Raf. An E-value of less than 0.05 indicates significant primary structure similarity between the proteins (7). According to the Dali server, the B-Raf kinase and C-Raf kinase share a Z-score of 36.3 for tertiary structure similarities. A Z-score higher than 2 means the proteins have similar tertiary structures (8). The kinase domain of C-Raf is located closer to the N-terminus in the protein than that of B-Raf (1). C-Raf serves the same biological role as B-Raf in the Ras-Raf-MEK-ERK signaling pathway; it is phosphorylated by Ras and phosphorylates MEK (1,2). Unlike B-Raf, C-Raf is not primed for activation. C-Raf requires a more complex phosphorylation and activation process by Ras due to the lack of negatively charged amino acids in the N-terminal of the kinase domain (1,9). 

      B-Raf kinase is activated by Ras phosphorylation of Thr-598 and Ser-601 in the catalytic cleft (1-3,6). These residues are located where the P loop and the activation segment come closer in the tertiary structure (1,6). The P loop is a glycine-rich nucleotide-binding region located in the small lobe, and the activation segment is in the catalytic cleft (1,2,4,6). The activation segment contains an intrinsically disordered fragment encompassing residues 600-611 (1,6,7). Oncogenic mutations in B-Raf tend to cluster around the P loop and the N-terminal side of the activation segment (1,2,4,6). 

       There are two main kinds of mutations in the B-Raf kinase domain that result in increased activation of MEK (1,3,6). Increasing the activation of MEK causes cell proliferation and tumor growth by interfering with the Ras-Raf-MEK-ERK signaling pathway (1-6,9). The less common type of mutation has reduced kinase activity, but activates MEK indirectly by activating C-Raf (1-4,6,9). C-Raf is normally activated by phosphorylation by Ras, like B-Raf. Mutant B-Raf can activate C-Raf independently of Ras because the proteins can form heterodimers (9). Mutations to Gly-465 and Gly-595 result in impaired kinase activity and indirect activation of MEK (1,6,9). 

       The majority of B-Raf kinase mutations mimic the phosphorylated form of B-Raf, including the most common B-Raf kinase domain mutation, the conversion of valine to glutamate at residue 599 (1-4,6). Glu-599 mutant B-Raf kinase activity is about 500 times greater than the non-mutant B-Raf kinase (1). The Val-599 to Glu-599 mutation accounts for over 90% of the B-Raf mutations in malignant melanomas. Though other mutations occur at residue 599, Glu-599 accounts for approximately 95% of the mutations at this residue. The overwhelming prevalence of this mutation is because it only requires a single DNA base substitution to change the protein amino acid (1,3,6). It is speculated that Val-599 to Glu-599 mutations may be an indirect consequence of UV exposure (1,3). 

      The B-Raf kinase domain is inactivated by the compound BAY43-9006 (PDB ID: BAX), also known as Sorafenib (1,4-6). Sorafenib is usually co-crystallized with the B-Raf kinase (1-6). Sorafenib contains three six-member aromatic carbon rings: the trifluoromethyl phenyl ring, the central ring, and the pyridyl ring (6). Sorafenib interacts with Lys-482, Glu-500, Leu-513, Thr-528, Trp-530, Cys-531 Phe-582, Asp-593, and Phe-594 of the B-Raf kinase domain (6). These residues are conserved in C-Raf, so Sorafenib can also inhibit C-Raf as well as other kinases (5,6). Sorafenib stabilizes the inactive conformation of the B-Raf kinase by associating the P loop with the activation segment (4,6). The inactive conformation cannot activate MEK, directly nor indirectly, even with a mutation, and therefore prevents cell proliferation. Sorafenib maintains the inactive conformation of the B-Raf kinase domain by occupying the site the kinase would occupy in the active, phosphorylated form. This is accomplished by the interactions between the trifluoromethyl phenyl ring on Sorafenib and Phe-594 on the B-Raf kinase (6). Sorafenib is marketed as Nexavar by Bayer and is FDA approved to treat unresectable hepatocellular carcinoma and advanced renal cell carcinoma.