Enzymology BRAF (gene)

1 enzymology

1.1 activation

1.1.1 relieving cr1 autoinhibition
1.1.2 cr3 domain activation


1.2 mechanism of catalysis

1.2.1 atp binding
1.2.2 phosphorylation


1.3 inhibitors

1.3.1 sorafenib
1.3.2 vemurafenib

enzymology

b-raf serine/threonine-specific protein kinase. such, catalyzes phosphorylation of serine , threonine residues in consensus sequence on target proteins atp, yielding adp , phosphorylated protein products. since highly regulated signal transduction kinase, b-raf must first bind ras-gtp before becoming active enzyme. once b-raf activated, conserved protein kinase catalytic core phosphorylates protein substrates promoting nucleophilic attack of activated substrate serine or threonine hydroxyl oxygen atom on γ-phosphate group of atp through bimolecular nucleophilic substitution.

activation
relieving cr1 autoinhibition

the kinase (cr3) domain of human raf kinases inhibited 2 mechanisms: autoinhibition own regulatory ras-gtp-binding cr1 domain , lack of post-translational phosphorylation of key serine , tyrosine residues (s338 , y341 c-raf) in cr2 hinge region. during b-raf activation, protein s autoinhibitory cr1 domain first binds ras-gtp s effector domain cr1 ras-binding domain (rbd) release kinase cr3 domain other members of human raf kinase family. cr1-ras interaction later strengthened through binding of cysteine-rich subdomain (crd) of cr1 ras , membrane phospholipids. unlike a-raf , c-raf, must phosphorylated on hydroxyl-containing cr2 residues before releasing cr1 become active, b-raf constituitively phosphorylated on cr2 s445. allows negatively charged phosphoserine repel cr1 through steric , electrostatic interactions once regulatory domain unbound, freeing cr3 kinase domain interact substrate proteins.

cr3 domain activation

after autoinhibitory cr1 regulatory domain released, b-raf s cr3 kinase domain must change atp-binding active conformer before can catalyze protein phosphorylation. in inactive conformation, f595 of dfg motif blocks hydrophobic adenine binding pocket while activation loop residues form hydrophobic interactions p-loop, stopping atp accessing binding site. when activation loop phosphorylated, negative charge of phosphate unstable in hydrophobic environment of p-loop. result, activation loop changes conformation, stretching out across c-lobe of kinase domain. in process, forms stabilizing β-sheet interactions β6 strand. meanwhile, phosphorylated residue approaches k507, forming stabilizing salt bridge lock activation loop place. dfg motif changes conformation activation loop, causing f595 move out of adenine nucleotide binding site , hydrophobic pocket bordered αc , αe helices. together, dfg , activation loop movement upon phosphorylation open atp binding site. since other substrate-binding , catalytic domains in place, phosphorylation of activation loop alone activates b-raf s kinase domain through chain reaction removes lid otherwise-prepared active site.

mechanism of catalysis

figure 2: base-catalyzed nucleophilic attack of serine/threonine substrate residue on γ-phosphate group of atp. step 1: chelation of secondary magnesium ion n581 , deprotonation of substrate ser/thr d576. step 2: nucleophilic attack of activated substrate hydroxyl on atp γ-phosphate. step 3: magnesium complex breaks down , d576 deprotonates. step 4: release of products.

to catalyze protein phosphorylation via bimolecular substitution of serine , threonine residues adp leaving group, b-raf must first bind atp , stabilize transition state γ-phosphate of atp transferred.

atp binding

b-raf binds atp anchoring adenine nucleotide in nonpolar pocket (yellow, figure 1) , orienting molecule through hydrogen-bonding , electrostatic interactions phosphate groups. in addition p-loop , dfg motif phosphate binding described above, k483 , e501 play key roles in stabilizing non-transferable phosphate groups. positive charge on primary amine of k483 allows stabilize negative charge on atp α- , β-phosphate groups when atp binds. when atp not present, negative charge of e501 carboxyl group balances charge.

phosphorylation

once atp bound b-raf kinase domain, d576 of catalytic loop activates substrate hydroxyl group, increasing nucleophilicity kinetically drive phosphorylation reaction while other catalytic loop residues stabilize transition state.(figure 2). n581 chelates divalent magnesium cation associated atp orient molecule optimal substitution. k578 neutralizes negative charge on γ-phosphate group of atp activated ser/thr substrate residue won t experience electron-electron repulsion when attacking phosphate. after phosphate group transferred, adp , new phosphoprotein released.

inhibitors

since constitutively active b-raf mutants commonly cause cancer (see clinical significance) excessively signaling cells grow, inhibitors of b-raf have been developed both inactive , active conformations of kinase domain cancer therapeutic candidates.

sorafenib

figure 3: b-raf kinase domain locked in inactive conformation bound bay43-9006. hydrophobic interactions anchor bay43-9006 in atp binding site while urea group hydrogen-bonding traps d594 of dfg motif. bay43-9006 trifluoromethyl phenyl ring further prohibits dfg motif , activation loop movement active confermer via steric blockage.

bay43-9006 (sorafenib, nexavar)is v600e mutant b-raf , c-raf inhibitor approved fda treatment of primary liver , kidney cancer. bay43-9006 disables b-raf kinase domain locking enzyme in inactive form. inhibitor accomplishes blocking atp binding pocket through high-affinity kinase domain. binds key activation loop , dfg motif residues stop movement of activation loop , dfg motif active conformation. finally, trifluoromethyl phenyl moiety sterically blocks dfg motif , activation loop active conformation site, making impossible kinase domain shift conformation become active.

the distal pyridyl ring of bay43-9006 anchors in hydrophobic nucleotide-binding pocket of kinase n-lobe, interacting w531, f583, , f595. hydrophobic interactions catalytic loop f583 , dfg motif f595 stabilize inactive conformation of these structures, decreasing likelihood of enzyme activation. further hydrophobic interaction of k483, l514, , t529 center phenyl ring increase affinity of kinase domain inhibitor. hydrophobic interaction of f595 center ring decreases energetic favorability of dfg conformation switch further. finally, polar interactions of bay43-9006 kinase domain continue trend of increasing enzyme affinity inhibitor , stabilizing dfg residues in inactive conformation. e501 , c532 hydrogen bond urea , pyridyl groups of inhibitor respectively while urea carbonyl accepts hydrogen bond d594 s backbone amide nitrogen lock dfg motif in place.

the trifluoromethyl phenyl moiety cements thermodynamic favorability of inactive conformation when kinase domain bound bay43-9006 sterically blocking hydrophobic pocket between αc , αe helices dfg motif , activation loop inhabit upon shifting locations in active conformation of protein.

vemurafenib

figure 4: structures of vemurafenib (right) , precursor, plx 4720 (left), 2 inhibitors of active conformation of b-raf kinase domain

plx4032 (vemurafenib) v600 mutant b-raf inhibitor approved fda treatment of late-stage melanoma. unlike bay43-9006, inhibits inactive form of kinase domain, vemurafenib inhibits active dfg-in form of kinase, firmly anchoring in atp-binding site. inhibiting active form of kinase, vemurafenib selectively inhibits proliferation of cells unregulated b-raf, cause cancer.

since vemurafenib differs precursor, plx4720, in phenyl ring added pharmacokinetic reasons, plx4720 s mode of action equivalent vemurafenib s. plx4720 has affinity atp binding site partially because anchor region, 7-azaindole bicyclic, differs natural adenine occupies site in 2 places nitrogen atoms have been replaced carbon. enables strong intermolecular interactions n7 hydrogen bonding c532 , n1 hydrogen bonding q530 preserved. excellent fit within atp-binding hydrophobic pocket (c532, w531, t529, l514, a481) increases binding affinity well. ketone linker hydrogen bonding water , difluoro-phenyl fit in second hydrophobic pocket (a481, v482, k483, v471, i527, t529, l514, , f583) contribute exceptionally high binding affinity overall. selective binding active raf accomplished terminal propyl group binds raf-selective pocket created shift of αc helix. selectivity active conformation of kinase further increased ph-sensitive deprotonated sulfonamide group stabilized hydrogen bonding backbone peptide nh of d594 in active state. in inactive state, inhibitor s sulfonamide group interacts backbone carbonyl of residue instead, creating repulsion. thus, vemurafenib binds preferentially active state of b-raf s kinase domain.