Phosphorylation of RAF Kinase Dimers Drives Conformational Changes that Facilitate Transactivation

Abstract RAF kinases are key players in the MAPK signaling pathway and are important targets for personalized cancer therapy. RAF dimerization is part of the physiological activation mechanism, together with phosphorylation, and is known to convey resistance to RAF inhibitors. Herein, molecular dynamics simulations are used to show that phosphorylation of a key N‐terminal acidic (NtA) motif facilitates RAF dimerization by introducing several interprotomer salt bridges between the αC‐helix and charged residues upstream of the NtA motif. Additionally, we show that the R‐spine of RAF interacts with a conserved Trp residue in the vicinity of the NtA motif, connecting the active sites of two protomers and thereby modulating the cooperative interactions in the RAF dimer. Our findings provide a first structure‐based mechanism for the auto‐transactivation of RAF and could be generally applicable to other kinases, opening new pathways for overcoming dimerization‐related drug resistance.

RAF kinases connect the Ras GTPase to activation of the MEK-ERK pathway.T his pathway regulates many fundamental cellular functions,i ncluding cell proliferation, and is dysregulated in approximately 50 %ofhuman cancers. [1] This pathway has thus been ak ey focus in cancer drug development. Arecent breakthrough came with the BRAF inhibitor vemurafenib,w hich achieved high response rates in BRAFmutated metastatic melanoma. [2] Interestingly,B RAF inhibition in RAS-mutated tumors induces paradoxical ERK activation and tumor progression owing to the formation of RAF dimers. [3] RAF dimerization is also amajor mechanism of acquired clinical resistance to RAF inhibitors. [4] Owing to its important clinical implications,R AF dimerization has attracted enormous interest. RAF homo-and heterodimers show significantly higher kinase activity than monomers,and it has been shown that physiological RAF activation involves dimerization. [1,5] Dimer activity remains high even when one protomer (denoted as the activator) is kinase-dead or inhibited, owing to allosteric transactivation of its binding partner (the receiver). [1,5] Recent data indicate that the Nterminal acidic (NtA) motif [6] is essential for the allosteric activation of RAF dimers. [7] This region is located just upstream of the kinase domain and mediates physiological activation. In RAF1, phosphorylation of the corresponding sequence SSYY (residues 338-341) is induced during RAF1 activation. [6b, 8] In BRAF (residues 446-449, sequence SSDD), the activating site S446 is constitutively phosphorylated and the tyrosines are replaced by negatively charged aspartates. This configuration of the NtA motif primes BRAF for activation, which may explain why single mutations of BRAF,s uch as V600E in the activation loop,c an cause full activation and drive cancer, while RAF1 mutations are rare in cancer. [9] There is no consensus on which kinase phosphorylates the NtA motif in vivo,s ince several kinases,i ncluding RAF1 itself,have been reported to be able to do this. [1,10] As shown by mutagenesis studies, [7] the NtA motif in the activator is required for transactivation of the receiver in RAF dimers.However,nostructural evidence is available to explain this allosteric activation process,since all RAF crystal structures lack the NtA motif.
Based on recently available crystallographic data for RAF (e.g.,P DB entries 4E26 [11] and 3OMV [3b] )w em odeled RAF homo-and heterodimers that include the NtA region and correspond to the smallest subset of residues present at the Nterminus of the kinase domain in constitutively dimerized, drug resistant splice variants [4] (Figure 1a). By using atomistic molecular dynamics (MD) simulations of kinase dimers, [12] we also investigated the role of phosphorylation, which is biochemically well documented but has eluded detailed structural studies.The computational modeling approach and the parameters used are described in the Methods section of the Supporting Information and summarized in Table S1.
Phosphorylation of the NtA motif generates several salt bridges that extend and stabilize the binding interface between two BRAF protomers ( Figure 1b). Intriguingly, these salt bridges are primarily interprotomer salt bridges, which are formed between the NtA motif and positive residues located either upstream of the NtA motif or at the C-terminal end of the aC-helix, the orientation of which plays an important role in kinase activation. [12a, 13] We note that all of the residues that form interprotomer salt bridges with phosphorylated residues of the NtA motif are conserved in the three RAF isoforms,b ut not in other kinases (Table S2 in the Supporting Information). Remarkably,t he C-terminal end of the aC-helix is neutral in most kinases,w hereas in RAFs it carries a + +3c harge.M oreover, mutations of these charged residues impair kinase activity [14] and affect RAF homo-and heterodimerization. [3b,14, 15] Since RAF kinases dimerize via the C-terminal end of the aC-helix, the accumulation of six positive charges at the dimerization interface enables interaction with the highly negatively charged NtA motif (specific to RAF), thereby promoting the dimerization. In fact, we estimate that NtA phosphorylation constitutes almost half of the interaction potential energy between the BRAF protomers ( Figure 2a and Table S1), predominantly by enhancing electrostatic interactions.Inparticular,weidentified two conserved Arg residues, R443 (R336) at the NtA region and R506 (R398) close to the aC-helix in BRAF (RAF1), that participate in the intermolecular salt bridges.R 506 (R398) has been reported previ-ously to play arole in dimerization, [14] and here we confirmed the relevance of R443 (R336) experimentally by co-immunoprecipitation, demonstrating ar educed dimerization propensity of R!Am utants (Figure 2b). Thed ifferences between BRAF and RAF1 in terms of their cellular phosphorylation states (i.e., BRAF is constitutively phosphorylated whereas RAF1 activation requires additional input [6b] ) and the exact sequences of the NtA motifs (Figure 1b,a nd Figures S1-S3 in the Supporting Information) explain the significant differences between the activities of BRAF and RAF1 homo-and heterodimers. [5c] Our simulations also reveal that the phosphorylated NtA motif is connected to the active site via the R-spine [16] (Figure 3a). This conserved hydrophobic structure connects four residues from critical sites in the kinase monomer, [16a,17] including the active site.T he R-spine is anchored to the aF-

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Communications 984 www.angewandte.org helix via ahydrogen bond between acarboxylate group at the N-terminal end of the aF-helix and the backbone of the HRD motif. [16b] An assembled R-spine indicates active kinase conformations,w hile ab roken R-spine correlates with inactive kinase conformations. [16] Previous experiments showed that mutation of ac onserved Tr pr esidue (W450 for BRAF,T able S2) impedes transactivation and impairs the dimerization of BRAF. [7] We observed that W450 extends the R-spine by forming stacking interactions with the fourth residue (R4, Figure 3a). Although W450 is highly conserved in most kinases (Table S2), the interaction between this Tr pa nd the R4 residue of the R-Spine is not always observed. R4 is aliphatic in about 80 %ofthe kinases,including PKA. In PKA, CDK2, and p38 structures,the R-spine and the corresponding Trpdo not interact. Although this Trpi sn ot conserved in EGFR, crystal structures show that its functional equivalent lysine (L680) [18] also interacts with the R-spine.R emarkably, mutation of L680 destabilizes the EGFR dimer in the active complex and impedes kinase activity. [18] We observed that the NtA motif together with W450 extends the dimerization interface,t hereby connecting the R-spines and thus the distant active sites of the two protomers.F or PKA, aliphaticto-aromatic mutation of R4 gives normal levels of catalytic activity. [19] This is also the case for BRAF F516A and F516L mutations,w hich maintain the catalytic activity (Figure 3b) because they preserve the integrity of the R-spine within the monomer.However,wefound that these BRAF mutants are unable to transactivate RAF1 in the heterodimer (Figure 3c). These results provide af irst structure-based explanation for the transactivation mechanism of RAF dimers following NtA motif phosphorylation.
Importantly,our results indicate phosphorylation-induced large-scale structural changes in RAF dimers,w hereas the unphosphorylated dimers remain structurally similar to their starting structures,i na greement with the crystallographic structures.O nly when all phosphorylated residues,i ncluding the two activation-loop residues near V600, are present in the simulated system, we observe large changes between the universally conserved HRD and DFG motifs (Figure 4). These changes provide insight into the structural flexibility Figure 3. a) R-spine of the two protomers of ATP-P BRAF. R1-R4 denotes the four residues that form the R-spine (H574, F595, L505, and F516 for BRAF). The conserved Trp(W450 for BRAF) extends the R-spine, connecting it with the dimerization interface. b) F516A mutation does not affect the activity of BRAF. HEK293T cells were transfected with FLAG-tagged BRAF mutants (as indicated).L ysates were collected the next day from growing cells. c) F516 is required for transactivation activity of kinase-dead BRAF. HEK293T cells were transientlyt ransfected with FLAG-FKBP-tagged BRAF and V5-FRBtagged RAF1. Heterodimerformation in serum-starved cells was induced by addition of 500 nm A/C heterodimerizer for 1hour.T otal lysates were collected and analyzed.S ubstitution of F516 in kinasedead BRAF K483M resulted in reduced ERK activation by RAF heterodimers. present in the kinase domains,and could provide astructural explanation for recent observations on transactivation. [7] Remarkably,t hese changes involve structural elements that are connected via the R-spine and/or the NtA motif,and they typically occur in only one of the protomers,t hereby giving rise to asymmetry in the dimer.I nterestingly,o nly structures that include vemurafenib or closely related inhibitors (PDB entries 3OG7, [20] 4FK3, [21] and 3C4C [21] )present asymmetrical dimers,which have been proposed as ageneral mechanism for the allosteric modulation of kinase activity. [22] Our protomer structures differ from these inactive crystal structures,s ince the presence of ATPenhances the stability of the salt bridge between K483 and E501, which is absent in the vemurafenibbound structures.Therefore,our protomer structures could be useful leads for the development of drugs that avoid the paradoxical kinase activation resulting from drug-induced RAF dimerization.
In summary,our results show that phosphorylation of the NtA motif promotes dimerization through several interprotomer salt bridges formed between the NtA motif of one of the protomers and the positively charged C-terminal end of the aC-helix of the other protomer.T hey further reveal the importance of the conserved tryptophan residue located at the N-terminal end of the kinase domain (W450 for BRAF), which plays ac rucial role by connecting the R-spines of the two protomers.T his suggests ac ooperative interprotomer interaction that is mediated by salt bridges involving the phosphorylated NtA motif.T he direct interaction of the Rspines via W450 explains why mutation of W450 abolishes the transactivation of RAF dimers. [7] More importantly,t his also explains how the phosphorylated dimers undergo significant conformational changes.T hese changes involve detachment of the R-spine from the anchoring aF-helix, and significant changes to the residue pair distances between the HRD and DFG motifs.T he asymmetric transactivation mechanism of RAF kinases also provides as tructural basis for understanding the paradoxical activation caused by type Ia nd type IIB inhibitors. [23]