CR3 BRAF (gene)

figure 1: inactive conformation of b-raf kinase (cr3) domain. p-loop (orange) hydrophobic interactions activation loop (gray) residues stabilize inactive kinase conformation shown sticks. f595 (red) blocks hydrophobic pocket atp adenine binds (yellow). d576 (orange) shown part of catalytic loop (magenta). figure modified pdb id 1uwh.

conserved region 3 (cr3), residues 457–717, makes b-raf s enzymatic kinase domain. largely conserved structure bi-lobal, connected short hinge region. smaller n-lobe (residues 457–530) responsible atp binding while larger c-lobe (residues 535–717) binds substrate proteins. active site cleft between 2 lobes, , catalytic asp576 residue located on c-lobe, facing inside of cleft.

subregions

p-loop

the p-loop of b-raf (residues 464–471) stabilizes non-transferable phosphate groups of atp during enzyme atp-binding. specifically, s467, f468, , g469 backbone amides hydrogen-bond β-phosphate of atp anchor molecule. b-raf functional motifs have been determined analyzing homology of pka analyzed hanks , hunter b-raf kinase domain.

nucleotide-binding pocket

v471, c532, w531, t529, l514, , a481 form hydrophobic pocket within adenine of atp anchored through van der waals attractions upon atp binding.

catalytic loop

residues 574–581 compose section of kinase domain responsible supporting transfer of γ-phosphate of atp b-raf s protein substrate. in particular, d576 acts proton acceptor activate nucleophilic hydroxyl oxygen on substrate serine or threonine residues, allowing phosphate transfer reaction occur mediated base-catalysis.

dfg motif

d594, f595, , g596 compose motif central b-raf s function in both inactive , active state. in inactive state, f595 occupies nucleotide-binding pocket, prohibiting atp entering , decreasing likelihood of enzyme catalysis. in active state, d594 chelates divalent magnesium cation stabilizes β- , γ-phosphate groups of atp, orienting γ-phosphate transfer.

activation loop

residues 596–600 form strong hydrophobic interactions p-loop in inactive conformation of kinase, locking kinase in inactive state until activation loop phosphorylated, destabilizing these interactions presence of negative charge. triggers shift active state of kinase. specifically, l597 , v600 of activation loop interact g466, f468, , v471 of p-loop keep kinase domain inactive until phosphorylated.