784 Biochemical Society Transactions (2014) Volume 42, part 4

Molecular mechanisms of asymmetric RAF dimeractivationPablo G. Jambrina*, Olga Bohuszewicz*, Nicolae-Viorel Buchete†, Walter Kolch‡§‖ and Edina Rosta*1

*Department of Chemistry, King’s College London, London SE1 1DB, U.K.

†School of Physics, University College Dublin, Dublin, Ireland

‡Systems Biology Ireland, University College Dublin, Dublin, Ireland

§Conway Institute, University College Dublin, Dublin, Ireland

‖School of Medicine and Medical Sciences, University College Dublin, Dublin, Ireland

AbstractProtein phosphorylation is one of the most common post-translational modifications in cell regulatorymechanisms. Dimerization plays also a crucial role in the kinase activity of many kinases, including RAF,CDK2 (cyclin-dependent kinase 2) and EGFR (epidermal growth factor receptor), with heterodimers oftenbeing the most active forms. However, the structural and mechanistic details of how phosphorylation affectsthe activity of homo- and hetero-dimers are largely unknown. Experimentally, synthesizing protein sampleswith fully specified and homogeneous phosphorylation states remains a challenge for structural biology andbiochemical studies. Typically, multiple changes in phosphorylation lead to activation of the same protein,which makes structural determination methods particularly difficult. It is also not well understood howthe occurrence of phosphorylation and dimerization processes synergize to affect kinase activities. In thepresent article, we review available structural data and discuss how MD simulations can be used to modelconformational transitions of RAF kinase dimers, in both their phosphorylated and unphosphorylated forms.

IntroductionPhosphate transfer and cleavage play essential roles in biolo-gical processes of all living organisms [1,2]. Phosphorylationis one of the most common post-translational modificationsof proteins [3] and is carried out by kinases that transfera phosphate group (-O-PO3

2 − ) from ATP to an aminoacid, with phosphorylation of serine, threonine or tyrosineresidues being the best studied reactions. The reversibleaddition of a phosphate group introduces two additionalnegative charges, which can trigger key conformationalchanges that alter the biological function of the protein.Thus phosphorylation can modulate the extent of enzymaticactivation or inactivation of kinases in signalling pathways,which transfer molecular signals from receptors on thecell membrane to intracellular effector molecules, includingtranscription factors in the nucleus [4–6].

One of the main signalling pathways that relate mito-genic signals involves ERKs (extracellular-signal-regulatedkinases). The ERK pathway consists of a chain of interactingproteins, RAS, RAF, MEK [MAPK (mitogen-activatedprotein kinase)/ERK kinase] and ERK, where the latterthree are kinases that transfer the signal via phosphorylation.

Key words: dimerization, extracellular-signal-regulated kinase signalling pathway (ERK signalling

pathway), molecular dynamics (MD), phosphorylation, RAF.

Abbreviations: AL, activation loop; CDK, cyclin-dependent kinase; CL, catalytic loop; EGFR,

epidermal growth factor receptor; ERK, extracellular-signal-regulated kinase; MEK, MAPK

(mitogen-activated protein kinase)/ERK kinase; NtA, N-terminal acidic; PKA, cAMP-dependent

protein kinase.1To whom correspondence should be addressed (email Edina.Rosta@kcl.ac.uk).

Importantly, this pathway is hyperactivated in >30 % ofall cancers, with RAS and RAF being the main oncogenicfactors [7]. ARAF, BRAF and CRAF (or RAF1) enzymesconstitute the RAF family of serine/threonine kinases. Single-residue mutations of BRAF, in particular V600E, are linkedto 66 % of malignant melanomas [8], and also to a largenumber of ovarian, colon and papillary thyroid cancers. TheV600E BRAF mutation is the most frequent cancer-causingprotein kinase mutation currently known [8]. A recentlyFDA (U.S. Food and Drug Administration)-approved RAFkinase inhibitor, vemurafenib, is highly effective against V600BRAF mutations in melanoma. However, the high efficacyof vemurafenib is offset by the rapid development of drugresistance, presenting acute clinical challenges [9].

Phosphorylation of several conserved residues is typicallyrequired for full activity of most kinases. However, despitethe prominent functional importance of phosphorylation inkinases such as RAFs, very limited structural and mechanisticinformation is available about activated phosphorylatedprotein complexes.

MD studies are often focused on the kinase domain, wherethe catalytic reaction takes place (Figure 1). In the kinasedomain, phosphorylation takes place in one or more residueswithin two conserved loops of the kinase domain in mostkinases: (i) the CL (catalytic loop), and (ii) the AL (activationloop). The AL has a specific sequence that is embraced withintwo strongly conserved sequence motifs: DFG (Asp-Phe-Gly) and APE (Ala-Pro-Glu), and its phosphorylation isrequired for activation in most kinases. RAF kinases are

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Figure 1 Main structural elements of BRAF dimers

(A) Phosphorylation sites of the extended BRAF kinase domain illustrated. The AL (dark blue), the CL (orange), the NtA

(red), the phosphorylated residues (sticks, labelled in black), and the αF- (yellow) and αC-helices (pink) are highlighted.

The bound ATP molecule (sticks, labelled in green) and the two Mg2 + ions (salmon spheres) were modelled on the basis

of the consensus PKA active-site architectures. (B) Conserved regions in RAF and EGFR kinase domains. The regulatory

phosphorylated residues are highlighted in squares.

also phosphorylated at the CL, which immediately precedesthe AL, starting with the universally conserved HRD (His-Arg-Asp) motif present in all kinases and ending with aconserved asparagine residue. Residues in these loops (e.g.Asn581 and Asp594 in BRAF) also contact the ATP and theMg2 + ions that serve as co-factors for phosphate transfer.In addition, active RAF kinases are also phosphorylated in

the so-called NtA (N-terminal acidic) motif [10] startingwith the 11th exon that immediately precedes the kinasedomain. Structural information on phosphorylated kinasesis available for only a few pathways. Producing samples ofhomogeneously phosphorylated proteins is experimentallychallenging, which is one of the main reasons for the lack ofcrystallographic structural information [11]. Several crystal

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786 Biochemical Society Transactions (2014) Volume 42, part 4

structures have been solved for the PKA (cAMP-dependentprotein kinase) proteins, for both their phosphorylated andunphosphorylated forms, and in complex with SP20, asubstrate-like peptide [12,13]. There are, however, only afew structures currently available of a kinase complexedwith its substrate kinase and, for all of them, the phospho-acceptor region is disordered. Only short model peptideshave been structurally characterized as kinase substrates insome cases, such as for PKA. For this case, the catalyticstep could thus be delineated to a greater detail [12].Unfortunately, experimental structural information is stillunavailable, although it is highly needed, owing to the crucialimportance of the protein kinase family both for future drugdevelopment and for current drugs in clinical use.

Dimerization of kinases: asymmetricactivationIt has been increasingly clear that dimerization and oligo-merization of kinases is also part of the biological activationmechanism of signalling pathways in vivo, characterizedby structural (and thus functional) asymmetry and co-operativity (e.g. [14]). Homo- and/or hetero-dimerizationare critically associated with the activation of kinases suchas EGFR (epidermal growth factor receptor), CDK (cyclin-dependent kinase) and RAF [15–20] and even of transcriptionfactors [21,22]. Assemblies of protein chains are also wellestablished to play key roles for AAA + (ATPases associatedwith various cellular activities) members [23]. These enzymescatalyse a closely related chemical reaction involving bondcleavage of the γ -phosphate which hydrolyses ATP to ADPand Pi. They also share some limited structural mechanisticsimilarities regarding ATP binding, such as the fact that theyalso co-ordinate Mg2 + ions that are required for the catalyticreaction, and that they use a phosphate-binding loop (P-loop or Walker-loop) motif to bind ATP (glycine-rich loop,residues 464–471 in BRAF) [24]. It remains a puzzle, however,how these molecular machines assemble and regulate theircatalytic activities. A key unanswered question is how themultiple active sites of the assembly carry out their catalyticactivities in a controlled sequential and co-operative manner.

RAF kinases have drawn significant attention dueto the recently developed melanoma drug vemurafenibthat is specifically active in BRAF mutant melanoma.Dimerization also plays a key role in the physiologicaland pathological regulation of RAF activity. Although RAFheterodimerization is transiently induced by growth factorstimulation, RAF inhibitors and mutated RAS enhance andsustain the formation of RAF dimers, leading to paradoxicalactivation of the ERK pathway or to clinical resistance againstRAF inhibitor drugs [25–29]. The structural basis for thesedramatic effects of RAF dimers are unknown. AlthoughRAF monomers are able to phosphorylate their substrateMEK, dimerization of RAF dramatically enhances kinaseactivity [30]. In particular, BRAF/CRAF heterodimers showthe highest activity in cells [30]. Interestingly, even homo-or hetero-dimerization of a kinase-competent RAF with

a kinase-dead RAF molecule increases the kinase activityof the dimer [18,30]. Similarly to the activation of EGFRfamily members, RAF forms asymmetric dimers, in whichone kinase domain (the activator) acts co-operatively asan allosteric activator of the other (the receiver) [31,32].Dimerization also plays key roles in drug action andin drug-resistance mechanisms [28]. Clinical studies haveshown that dimerization of aberrantly spliced BRAF V600Econtaining residues from the 11th exon, i.e. essentially fromthe NtA motif, is a significant source of resistance tovemurafenib [29]. Intriguingly, the dimerization-dependentactivation is enhanced by ATP-competitive inhibitors suchas vemurafenib [9]. Some RAF inhibitors can activate wild-type RAF proteins in a dimerization-dependent mechanism,known as the ‘paradoxical activation of RAF’, and cause sideeffects in clinical use [26]. Recent work proposed that theparadoxical activation of the drugs is due to impeding aninhibitory autophosphorylation at the P-loop [33], whichdoes not occur in mutant BRAF. However, if ATP ispresent, but not the substrate MEK, wild-type RAFis autoinhibited via phosphorylation on the P-loop. Wild-type BRAF is thereby activated if the bound drug inhibitsits autophosphorylation function. Importantly, activation ofthe RAF catalytic phosphorylation function is linked tofunctionally asymmetric dimers (i.e. one protomer stimulatesthe activity of its partner) both for MEK phosphorylation andfor inhibitory autophosphorylation. Interestingly, however,there is a lack of structural explanation for the asymmetryin RAF kinases. Most RAF dimer structures are foundto be essentially symmetrical. Only a few structures withvemurafenib-like inhibitors bound (PDB codes 3OG7 [34],4FK3 [35], 3C4C [35]) offer insight into the structuralasymmetry. Yet, a detailed structural elucidation is lackingthat could explain the functional asymmetry of the protomersobserved in RAF dimers.

Activation-related conformational changesKinases have several biologically relevant inactive and activeconformations [31,36–39]. Conformational states are oftenstabilized by protein-binding partners via the assembly ofbiologically active complexes and/or by the appropriatephosphorylation of the appropriate residues. Two mainconformational motions have been identified: (i) ‘DFG-in’ and ‘DFG-out’ conformations [35], and (ii) ‘αC-helixin’ and ‘αC-helix out’ conformations [36,39]. In the DFG-out (inactive) conformation, the phenylalanine residue flipsinto the active site, preventing ATP binding, whereas only theDFG-in (active) conformation can accommodate ATP inthe active site [11]. The αC-helix shift has been characterizedin several kinases, such as EGFR, CDK and also RAF [17,39].In RAF, it is typically associated with specific inhibitors thatpreferably bind to the tilted conformation [9]. However, itis not well understood what triggers the αC-helix shiftingfor the biologically relevant mechanism in the absenceof inhibitors. For RAF, phosphorylation is potentiallyresponsible for driving the conformational change of the

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Figure 2 Activation-related conformational changes in kinases

(A) Illustration of the angle ω characterizing the tilt of the αC-helix (pink) in BRAF. ω is defined by the positions of

three Cα atoms (C1, C2 and C3, red spheres). C1 is in the C-terminus of the αC-helix, C2 is part of a central residue,

usually alanine or serine, of the ‘anchor’ αF-helix (yellow), and C3 is from the N-terminus of the αC-helix, preceding

C1 by 12 residues. The active-site ATP molecule (sticks, coloured as in Figure 1), Asp594 (sticks, part of the DFG motif)

and two Mg2 + ions (pink spheres) are also shown. (B) Distribution of ω values measured for all crystal structures

of six kinases (PKA, MEK1, ERK2, BRAF, CDK2 and EGFR) currently available in the PDB (see Supplementary Figure S1

at http://www.biochemsoctrans.org/bst/042/bst0420784add.htm). (C) Multiple salt bridges (broken lines) stabilize the

inter-protomer contacts in the BRAF homodimer, with Ser446 of the NtA motif phosphorylated.

αC-helix shift during the biological activation mechanism.Phosphorylation of the NtA motif in RAF dimers has beenassociated with activation and enhanced dimerization [40].It has been shown recently that phosphorylation at thisnormally constitutively phosphorylated site is required toobserve the allosteric activation and functional asymmetryof RAF kinase dimers [32]. Interestingly, the so-calledjuxtamembrane segment has been shown to provide this rolein the EGFR family [16]. This segment is located immediatelypreceding the kinase domain, analogously to the location of

the NtA motif for RAF proteins. An additional similaritybetween these two kinase families is presented with the keyrole of a pseudokinase that forms dimers with the activekinases. KSR (kinase suppressor of RAS) serves this role inthe ERK pathway [41], whereas ErbB3 [or HER3 (humanepidermal growth factor receptor 3)] heterodimerizes withErbB1, ErbB2 and ErbB4 to modulate signal transduction[31]. In this respect, CRAF resembles ErbB2, which haslow activity as a monomer or heterodimer; however, itheterodimerizes with ErbB1 and ErbB4, and these dimers

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788 Biochemical Society Transactions (2014) Volume 42, part 4

constitute the main activity of signal processing in cells [42].Despite the newly available mutation experiments elucidatingthe role of phosphorylation at the NtA motif, structuraljustification underlying the activation mechanism remainselusive. It is yet to be investigated what are the correspondingactive and inactive states, and what is the role of the αC-helixin this allosteric activation mechanism.

We analysed the αC-helix angle tilt in a diverse set ofkinases. The αC-helix angle (ω) was defined using three α-carbon atom co-ordinates (C1, C2 and C3) (Figure 2A). Fordefining the positions of C1 and C3, we selected the Cα atomsof two residues: C1 at the C-terminal end of the αC-helix andC3 that precedes it by 12 amino acids, at the N-terminal endof the αC-helix. The position of the angle’s vertex (atom C2)(Figure 2A) was placed at the centre of the αF-helix. Theamino acids used for each kinase are listed in Supplement-ary Table S1 (at http://www.biochemsoctrans.org/bst/042/bst0420784add.htm). This conserved helix motif is knownto form a stable anchor, which supports the kinase activesite and the remaining kinase structural motifs [43]. MDsimulations also demonstrated that there is an effectivecommunication between this αF-helix, which plays an‘integrating’ role, and the more mobile ‘mediating’ αC-helixshown to be important for allosteric activation [44]. Theexact choice of the selected residue for the vertex did notsignificantly affect the observed αC-helix angle and resultedin essentially the same distribution. Several kinases, suchas BRAF, CDK2, EGFR and the insulin receptor kinasehave shown a bimodal distribution of the αC-helix ω angle,whereas others such as PKA, PDK1 (phosphoinositide-dependent kinase 1) and p38 kinase were observed in onlyone selected angle (Figure 2B, and Supplementary Figure S1at http://www.biochemsoctrans.org/bst/042/bst0420784add.htm).

Interestingly, for the kinases that show bimodal ω

distributions, there is also a good correlation between ω andthe distance between the lysine residue of the conservedVA[I/V]K (Val-Ala-Ile/Val-Lys) motif and a conservedglutamic acid residue in the centre of the αC-helix angle (Sup-plementary Figure S2 at http://www.biochemsoctrans.org/bst/042/bst0420784add.htm). For larger ω angles, a salt bridgeforms between these two residues. The salt bridge can orientthe ATP in the active-site cleft between the N- and the C-lobes, which is considered a prerequisite for active kinases[11].

Atomistic MD studies of kinaseconformational dynamicsComputational modelling methods such as atomistic MDstudies are becoming mainstream tools for complementingexperiments in building atomistic structural models ofbiological complexes, as recognized by the 2013 Nobel Prizein Chemistry [45]. Several computational studies observedconformational dynamics in kinases leading not only tostructures also supported by X-ray crystallographic data, butalso to novel structurally well characterized states [46].

Long-time MD simulations of membrane-embeddedEGFR suggest that, on one hand, ligand binding inducesdimerization of the juxtamembrane segments and formationof activated asymmetric kinase dimers [46]. On the otherhand, ligand-free dimers favour juxtamembrane segmentdissociation and membrane burial, and formation of inactivesymmetric kinase dimers [46]. Enhanced sampling methods,such as metadynamics, were also used to elucidate andcharacterize active and inactive states [47]. Wild-type andmutant forms of EGFR showed conformational differencesof the salt bridges between the VAIK motif, the αC-helix andthe AL, affecting the stabilization of the active forms of thekinase [47]. The phospho-transfer reaction between ATP andKemptide (a small heptameric synthetic peptide), catalysedby PKA, has also been studied using QM/MM (quantummechanics/molecular mechanics) methods [48]. Interestingly,these results show that the catalytic mechanism depends onthe phosphorylated state of the kinase [48].

Recent MD simulations (P.G. Jambrina, N. Rauch, K.Rybakova, N.-V. Buchete, W. Kolch and E. Rosta, unpub-lished work) suggest that asymmetric shifting of the αC-helixmay be facilitated by phosphorylation in RAF dimers. Weobserved strong ionic interactions between the protomersdue to phosphorylation of the NtA motif (Figure 2C). Theunderlying structures also provide explanation of mutationalexperiments regarding the role of Trp450. Our results afford astructural explanation for the recent experimental evidencethat phosphorylation of the NtA motif is required forasymmetric allosteric activation in BRAF homodimers andBRAF–CRAF heterodimers [32]. The observed activationmechanism from MD simulations in RAF thus may provideinsights into a more general activation mechanism used insignalling pathways.

Long-time MD simulations have the capability to provideunique insights into atomistically detailed structural data ofexperimentally difficult to observe conformational changesthat govern the activation processes. Atomistic simulationscould be particularly useful for predicting new structures inthe presence of the additional negative charges presented bythe phosphorylated residues [49]. For many kinase studies,homogeneous phosphorylation of specific protein residues isoften experimentally challenging, although it could be easilyachieved using carefully designed atomistic MD simulations.Computational modelling and MD studies thus also offerunique starting points and approaches, highly needed forkinases, in the search for new drugs, and can provide insightsinto experimental structure determination and biochemicalmeasurements.

Acknowledgements

We thank Nora Rauch and Katya Rybakova for their useful

discussions. E.R. acknowledges computational support from the

National Institutes of Health Biowulf cluster, and N.-V.B. from the

Irish Centre for High-End Computing (ICHEC).

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Signalling and Acquired Resistance to Targeted Cancer Therapeutics 789

Funding

W.K. is supported by Science Foundation Ireland [grant number

06/CE/B1129].

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Received 4 February 2014doi:10.1042/BST20140025

C©The Authors Journal compilation C©2014 Biochemical Society

Biochemical Society Transactions (2014) Volume 42, part 4

SUPPLEMENTARY ONLINE DATA

Molecular mechanisms of asymmetric RAF dimeractivationPablo G. Jambrina*, Olga Bohuszewicz*, Nicolae-Viorel Buchete†, Walter Kolch‡§‖ and Edina Rosta*1

*Department of Chemistry, King’s College London, London SE1 1DB, U.K.

†School of Physics, University College Dublin, Dublin, Ireland

‡Systems Biology Ireland, University College Dublin, Dublin, Ireland

§Conway Institute, University College Dublin, Dublin, Ireland

‖School of Medicine and Medical Sciences, University College Dublin, Dublin, Ireland

Figure S1 Distribution of ω values measured for all crystal structures of the selected kinases studied

See Table S1 for more details.

1To whom correspondence should be addressed (email Edina.Rosta@kcl.ac.uk).

C©The Authors Journal compilation C©2014 Biochemical Society Biochem. Soc. Trans. (2014) 42, 784–790; doi:10.1042/BST20140025

Signalling and Acquired Resistance to Targeted Cancer Therapeutics

Figure S2 Correlation between ω and the salt bridge distance between the VAVK motif lysine residue and the conserved glutamic acid

residue at the centre of the αC-helix

Data show the results using all currently available crystal structures for the kinases analysed. See Table S1 for more details.

C©The Authors Journal compilation C©2014 Biochemical Society

Biochemical Society Transactions (2014) Volume 42, part 4

Table S1 A diverse set of kinases with large numbers of available crystallographic structures were aligned

Residues corresponding to the Cα atoms used as C1, C2 and C3 in the definition of the ω angle (Figure 2A of the main text) are given. The corresponding

UniProt (http://www.uniprot.org) codes and the number of protomers used in the histograms (Figure 2B of the main text, and Figures S1 and S2)

are also given. Abbreviations: IGF1, insulin-like growth factor 1; MAPK, mitogen-activated protein kinase; PDK1, phosphoinositide-dependent kinase

1; ZAP70, ζ -chain (T-cell receptor)-associated protein kinase of 70 kDa. Organisms: B. taurus, Bos taurus (cow); H. sapiens, Homo sapiens (human);

M. musculus, Mus musculus (mouse).

Residues used to define ω

Kinase name Organism UniProt code C1 C2 C3 Number of protomers

PKA H. sapiens P17612 Val98 Ala223 Glu86 180

B. taurus P00517

M. musculus P05132

Akt1 H. sapiens P31749 Ser205 Gly334 Ala193 15

Aurora A kinase H. sapiens O14965 Leu188 Ser314 His176 95

BRAF H. sapiens P15056 Thr508 Ala641 Gln496 65

Casein kinase 1 H. sapiens P48730 Met59 Ser198 Pro47 23

CDK2 H. sapiens P24941 Leu58 Ser188 Ser46 418

EGFR H. sapiens P00533 Val745 Ser875 Lys733 80

ERK2 H. sapiens P28482 Phe78 Ser213 Gln66 38

IGF1 receptor H. sapiens P08069 Phe1027 Ser1167 Ile1015 40

Insulin receptor H. sapiens P06213 Phe1054 Ser1194 Ile1942 20

Lck H. sapiens P06239 Leu295 Ser425 Ala284 35

MEK1 H. sapiens Q02750 Cys121 Ser248 Asn109 34

p38 MAPK H. sapiens Q16539 Met78 Ser208 Lys66 217

PDK1 H. sapiens O15530 Arg136 Ala267 Val124 58

ZAP70 H. sapiens P43403 Leu393 Ser524 Glu381 3

Received 4 February 2014doi:10.1042/BST20140025

C©The Authors Journal compilation C©2014 Biochemical Society