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The complex of wild type B-RAF and BAY439006

PDB ID 1uwh

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1uwh, resolution 2.95Å ()
Ligands: ,
Related: 1uwj
Resources: FirstGlance, OCA, RCSB, PDBsum
Coordinates: save as pdb, mmCIF, xml


The complex of wild type B-RAF and BAY439006[1] (1uwh)


IntroductionIntroduction

The B-RAF Protein belongs to a large family of protein kinases that have a very important role on different aspects of cellular biology and biomedical sciences. The human protein kinase family consists of 518 genes, making it one of the largest gene families[2]. Based upon the phosphorylation of the hydroxyl group these proteins are classified on 3 groups: protein serine threonine kinase (385 members), protein tyrosine kinase (90 members) and tyrosine kinase like proteins (43 members). A-RAF, B-RAF and C-RAF (3omv) proteins are a family of serine threonine kinase proteins that participate in the RAS-RAF-MEK-ERK signal transduction cascade. The general reaction they catalyse is:

This cascade participates in the regulation of several cellular mechanisms like: apoptosis, cell cycle progression, differentiation, proliferation and transformation to the cancerous state in response to growth factors, cytokines and hormones. First discovered in 1938[3] as a retroviral oncogene B-RAF plays a key role on all studies concerning cancer therapies and other biomedical applications.


B-RAF Structure[4]B-RAF Structure[4]

3D View of the complex ; resolution 2,95 A

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As we distinguished before, there are 3 types of RAF proteins: A-RAF, B-RAF and C-RAF (3omv). All of them share 3 much conserved regions (CR): CR1, CR2 and [5]. We have to say here that in the pdb file 1UWH the protein is already dimerized (as we will say it in the next part), and so we have here two CR3 domains.
CR1 is composed of a RAS binding domain (RBD) and a cysteine rich domain (CRD) which can bind 2 zinc ions (zinc finger domain). It is possible for the CR1 to interact with the RAS and with the membrane phospholipids (PH domain).
CR2 is a serine threonine rich domain. When a serine is phosphorylated this domain can bind a regulatory protein that can bind the C-terminal region in the same time. Binding of 14-3-3 (C-terminal region) to this phosphorylated serine is inhibitory for the enzyme.
is the protein kinase domain on the C-terminal region. We can find just after this kinase domain a stimulatory 14-3-3 binding site for other regulatory proteins.

Diagram of the inferred interactions between human B-RAF kinase catalytic core residues, ATP, and MEK[6]

As all RAF Proteins, B-RAF has the characteristic and the . The small lobe is constituted of an antiparallel β-shift that anchors and orient the ATP. It contains a glycine rich ATP phosphate binding loop, called . The large lobe interacts with the substrate that in our case is MEK1/2 who needs to be phosphorylated to be active. The [7] is just between the two lobes. These 2 lobes can move relative to each other, opening or closing the cleft. This has 2 major consequences on the functioning of this enzyme:

1. The open form allows access of ATP and release of ADP from the active site.

2. The closed form brings the residues of the substrate into the active site.

On the other hand each lobe has her proper polypeptide segment that can change conformation from active to inactive and vice versa.

1. In the small lobe, this segment is a α-helix, which is called αC-helix. The αC-helix rotates and translates with respect to the rest of the lobe, making or breaking part of the active site.

2. In the large lobe, the activation segment can make or break part of the ATP binding site[8].

The activation segment of each protein kinase has a specific domain that begins with a DFG amino acid sequence that can easily change its conformation (active or inactive conformation). In the inactive conformation[9] the phenylalanine side chain occupies the ATP binding pocket and the aspartate side chain faces away from the active site. This is the DFG Aspartate Out Conformation. In the active conformation, the phenylalanine side chain is rotated out of the ATP binding pocket and the Aspartate side chain can now face the ATP binding pocket and form coordinated links with the Mg²⁺. This is called the DFG Aspartate In Conformation. The activation segment can be phosphorylated by members of the same protein kinase family or by other protein kinases.
A separates the adenine binding site from the hydrophobic pocket. Mutation on this residue can prevent the binding of kinase inhibitory drugs (the replacement of a threonine by a methionine for example).

A. Structure of B-RAF Kinase Domain
B. Schematic B-RAF Primary Structure[10]

It is very important to distinguish conserved amino acid residue signatures that constitute the catalytic core of the B-RAF Kinase. The catalytic properties are done by the Motif and the starts in general with a and ends with . But how the catalytic reaction is generated? An invariant lysine ( in B-RAF) forms salt bridges with the gamma phosphate of the ATP. Asp576, which is a base in the catalytic loop, orients the seryl or threonyl group of the substrate protein and takes the proton of the hydroxyl group, facilitating the attack of oxygen on the gamma phosphorus atom of MgATP. Asp594 binds Mg²⁺ which coordinates the beta and gamma phosphates of ATP.


This is a table of the important residues of B-RAF:

Domain    Residue
RBD:    155-227
CRD:    234-280
CR1:    150-290
CR2:    360-375
:    451-717
[11]:    463-471
14-3-3 binding site:    S365-S729
:    T529
:    574-576
of :    578
:    594-596
Activation segment phosphorylation sites:    -S602
End of Activation segment:    621-623
No. of residues:    766
Molecular Weight (kDa):    84.4
UniProtKB accession No.:    P15056


Regulation[12]Regulation[12]

B-RAF in the MAP Kinase PathwayB-RAF in the MAP Kinase Pathway

Diagram of the RAS-RAF-MEK-ERK pathway[13].

The regulation of RAF Kinases involves protein-protein interactions, phosphorylations, dephosphorylations and conformational changes[14].
RAF kinases participate in the RAS-RAF-MEK-ERK signal transduction cascade, which is sometimes denoted as the mitogen-activated protein kinase (MAPK) cascade[15]. Growth factors bind to receptor tyrosine kinases (RTKs), resulting in RAS activation. RAF proteins are one of a family of effector proteins activated by RAS, and they in turn stimulate the activation of MEK, which subsequently stimulates ERK activity. ERK phosphorylates both cytosolic and nuclear proteins, thereby mediating the cellular responses cells make when this pathway is activated.

Structural implication on the MAP Kinase PathwayStructural implication on the MAP Kinase Pathway

Under non stimulatory conditions a serine of CR2 domain and another near the C-terminus are phosphorylated and are bound to 14-3-3 domain. To activate RAF Kinases, RAS-GTP has to interact with the RDB Domain. This is necessary but not enough to activate the RAF Kinase. One characteristic that distinguish B-RAF from A or C-RAF is the presence of Asp448 and Asp449 on the N-terminal region, which bear negative charges. Phosphorylation of and Ser602 in the activation segment is essential for B-RAF activation. That’s why the basal activity of B-RAF is greater than A or C-RAF (3omv).

Although X-ray structures of B-RAF kinase domains were said to be monomeric, each of the six structures in the protein data bank (2009), including B-RAF (V600E), showed that the asymmetric unit of the crystal contains two kinase domains that interact in a unique side-to-side fashion involving the N-lobe[16]. This side-to-side dimerization involves the αC helix, an important determinant of active and inactive conformations. Based upon ultracentrifugation studies, it is demonstrated that the catalytic domain of human B-RAF forms homodimers. Arg509 of B-RAF occurs in the side-to-side interface, and these investigators found that the Arg509[17] His mutant exists as a monomer. But B-RAF can form heterodimers too[18]. Ser621 and residues in the αC helix of C-RAF (3omv) participate in dimer formation. B-RAF–C-RAF heterodimers are more active than either homodimer. Phosphorylation of B-RAF at Thr753 as catalyzed by ERK destabilizes heterodimer formation with C-RAF and decreases kinase activity.


The RAF Inhibitor Paradox[19]The RAF Inhibitor Paradox[19]

RAF kinase inhibitors effectively block MEK and ERK phosphorylation. However the B-RAF specific inhibitor 885-A produces an unexpected increase in ERK phosphorylation in human melanoma cell lines. How can a RAF kinase inhibitor lead to the paradoxical increase in RAF kinase activity and ERK phosphorylation? Two additional studies noted below address this issue, and the common finding is that the binding of inhibitors to RAF kinases promotes RAS-dependent C-RAF homo- or heterodimerization and C-RAF activation.

3D View of C-RAF

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First StudyFirst Study

In a first study Heidorn and al. blocked N-RAS and C-RAF by RNA interference. The result showed that the ERK activity was blocked, indicating that RAS and C-RAF participate in the paradoxical response. They reported that 885-A binding to B-RAF or mutation to a kinase-dead B-RAF drives their binding to C-RAF. The introduction of a gatekeeper mutation in B-RAF abolishes the ability of B-RAF inhibitors to induce the binding of B-RAF to C-RAF (3omv). Inhibitor binding to B-RAF in the presence of activated RAS induces B-RAF binding to C-RAF leading to C-RAF activation and increased downstream ERK phosphorylation and activation.

Second StudySecond Study

In a second study, Poulikakos and al. reported that six ATP-competitive RAF inhibitors induce ERK activation in cells with activated RAS and wild-type B-RAF but inhibit signaling in activated mutant B-RAF cells[20]. B-RAF and C-RAF form homo- and heterodimers following RAS activation. PLX4032 and PLX4720, RAF kinase inhibitors, induce the phosphorylation of MEK and ERK in wild-type and B-RAF -/- mouse embryonic fibroblasts. The response is diminished in C-RAF -/- fibroblasts, arguing on the importance of C-RAF in paradoxical MEK-ERK activation.

Third StudyThird Study

In a third study, Hatzivassiliou and colleagues reported that RAF inhibitor treatment results in the paradoxical induction of phospho-MEK and phospho-ERK levels in the wild-type RAS/RAF human Melanoma[21]. They showed that knockdown of C-RAF, but not B-RAF, in human colorectal carcinoma HCT116 (mutant K-RAS) cells reverses the phospho-MEK induction observed after RAF inhibitor treatment, indicating that C-RAF has a major role in signaling to MEK, a result that is in agreement with the above two studies.

These studies indicate that the binding of an inhibitor to C-RAF leads to the formation of a C-RAF homodimer and C-RAF activation resulting in downstream MEK-ERK activation. Another possible, but not mutually exclusive, mechanism is that binding of an inhibitor to B-RAF leads to the formation of a B-RAF–C-RAF heterodimer and C-RAF activation. That RAF-kinase-induced paradoxical activation occurs in B-RAF -/- mouse embryonic fibroblasts does not rule out the possibility that B-RAF–C-RAF heterodimers play a role in paradoxical activation. A recent study showed that A-RAF acts as a Scaffold to stabilize the heterodimers of B-RAF and C-RAF[22].


B-RAF in cancersB-RAF in cancers

RAS mutations occur in 15–30% of all human cancers, and B-RAF mutations occur in 30–60% of melanomas, 30–50% of thyroid cancers, and 5–20% of colorectal cancers. B-RAF mutants occur in a variety of cancers while mutants of the other two RAF enzymes in cancers are very rare. The majority of B-RAF mutations occur in the activation segment or in the glycine-rich loop. These mutations disrupt the inactive state to favor the active state. A Val600Glu mutation is one of the more known B-RAF mutations. This mutation occurs within the activation segment where the introduction of negative charges favors the formation of an active conformation. The introduction of glutamate into the activation segment of C-RAF fails to produce an activated enzyme most likely owing to the need for a negatively charged N-region for activity.

RAF kinases are attractive cancer drug targets. Pre-clinical studies with cell lines and tumor xenographs bearing B-RAF mutations indicate that RAF kinase inhibitors are effective in decreasing cell proliferation. PLX4032, which has higher affinity for B-RAF than wild-type B-RAF, inhibits cancer progression in several animal models. The compound has demonstrated efficacy in Phase I clinical[23] trials in the treatment of melanoma patients. Sorafenib has a lower affinity for B-RAF than C-RAF and is ineffective as monotherapy in the treatment of melanoma. RAF kinase inhibitor treatment of cancers with wild-type or activated mutant RAS co-expressed with wild-type B-RAF may be deleterious owing to up-regulation of RAF kinase signaling. Deciphering the mechanisms of RAS-RAF-MEK-ERK signaling continues to be an important and challenging task.


External RessourcesExternal Ressources

PDB file for 1UWH
PDB file for 3OMV


ReferencesReferences

  1. ↑ Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Jones CM, Marshall CJ, Springer CJ, Barford D, Marais R. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004 Mar 19;116(6):855-67. PMID:15035987
  2. ↑ Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science. 2002 Dec 6;298(5600):1912-34. PMID:12471243 doi:10.1126/science.1075762
  3. ↑ Zebisch A, Troppmair J. Back to the roots: the remarkable RAF oncogene story. Cell Mol Life Sci. 2006 Jun;63(11):1314-30. PMID:16649144 doi:10.1007/s00018-006-6005-y
  4. ↑ Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Jones CM, Marshall CJ, Springer CJ, Barford D, Marais R. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004 Mar 19;116(6):855-67. PMID:15035987
  5. ↑ Wellbrock C, Karasarides M, Marais R. The RAF proteins take centre stage. Nat Rev Mol Cell Biol. 2004 Nov;5(11):875-85. PMID:15520807 doi:10.1038/nrm1498
  6. ↑ Roskoski R Jr. ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol Res. 2012 Aug;66(2):105-43. doi: 10.1016/j.phrs.2012.04.005. Epub 2012 , Apr 27. PMID:22569528 doi:10.1016/j.phrs.2012.04.005
  7. ↑ Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42-52. PMID:3291115
  8. ↑ Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42-52. PMID:3291115
  9. ↑ Seeliger MA, Ranjitkar P, Kasap C, Shan Y, Shaw DE, Shah NP, Kuriyan J, Maly DJ. Equally potent inhibition of c-Src and Abl by compounds that recognize inactive kinase conformations. Cancer Res. 2009 Mar 15;69(6):2384-92. Epub 2009 Mar 10. PMID:19276351 doi:10.1158/0008-5472.CAN-08-3953
  10. ↑ Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Jones CM, Marshall CJ, Springer CJ, Barford D, Marais R. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 2004 Mar 19;116(6):855-67. PMID:15035987
  11. ↑ Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR, Futreal PA. Mutations of the BRAF gene in human cancer. Nature. 2002 Jun 27;417(6892):949-54. Epub 2002 Jun 9. PMID:12068308 doi:10.1038/nature00766
  12. ↑ Roskoski R Jr. ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol Res. 2012 Aug;66(2):105-43. doi: 10.1016/j.phrs.2012.04.005. Epub 2012 , Apr 27. PMID:22569528 doi:10.1016/j.phrs.2012.04.005
  13. ↑ Dhomen N, Marais R. New insight into BRAF mutations in cancer. Curr Opin Genet Dev. 2007 Feb;17(1):31-9. PMID:17208430 doi:10.1016/j.gde.2006.12.005
  14. ↑ Wellbrock C, Karasarides M, Marais R. The RAF proteins take centre stage. Nat Rev Mol Cell Biol. 2004 Nov;5(11):875-85. PMID:15520807 doi:10.1038/nrm1498
  15. ↑ Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science. 2002 Dec 6;298(5600):1912-34. PMID:12471243 doi:10.1126/science.1075762
  16. ↑ Rajakulendran T, Sahmi M, Lefrancois M, Sicheri F, Therrien M. A dimerization-dependent mechanism drives RAF catalytic activation. Nature. 2009 Sep 24;461(7263):542-5. doi: 10.1038/nature08314. Epub 2009 Sep 2. PMID:19727074 doi:10.1038/nature08314
  17. ↑ Rajakulendran T, Sahmi M, Lefrancois M, Sicheri F, Therrien M. A dimerization-dependent mechanism drives RAF catalytic activation. Nature. 2009 Sep 24;461(7263):542-5. doi: 10.1038/nature08314. Epub 2009 Sep 2. PMID:19727074 doi:10.1038/nature08314
  18. ↑ Rushworth LK, Hindley AD, O'Neill E, Kolch W. Regulation and role of Raf-1/B-Raf heterodimerization. Mol Cell Biol. 2006 Mar;26(6):2262-72. PMID:16508002 doi:10.1128/MCB.26.6.2262-2272.2006
  19. ↑ Roskoski R Jr. ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol Res. 2012 Aug;66(2):105-43. doi: 10.1016/j.phrs.2012.04.005. Epub 2012 , Apr 27. PMID:22569528 doi:10.1016/j.phrs.2012.04.005
  20. ↑ Poulikakos PI, Zhang C, Bollag G, Shokat KM, Rosen N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature. 2010 Mar 18;464(7287):427-30. doi: 10.1038/nature08902. PMID:20179705 doi:10.1038/nature08902
  21. ↑ Heidorn SJ, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I, Dhomen N, Hussain J, Reis-Filho JS, Springer CJ, Pritchard C, Marais R. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell. 2010 Jan 22;140(2):209-21. doi: 10.1016/j.cell.2009.12.040. PMID:20141835 doi:10.1016/j.cell.2009.12.040
  22. ↑ Collier R. Professionalism: the importance of trust. CMAJ. 2012 Sep 18;184(13):1455-6. doi: 10.1503/cmaj.109-4264. Epub 2012 Aug 27. PMID:22927515 doi:10.1503/cmaj.109-4264
  23. ↑ Pratilas CA, Solit DB. Targeting the mitogen-activated protein kinase pathway: physiological feedback and drug response. Clin Cancer Res. 2010 Jul 1;16(13):3329-34. doi: 10.1158/1078-0432.CCR-09-3064., Epub 2010 May 14. PMID:20472680 doi:10.1158/1078-0432.CCR-09-3064


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