Recognition of BRAF by CDC37 and Re-Evaluation of the Activation Mechanism for the Class 2 BRAF-L597R Mutant

Abstract

The kinome specific co-chaperone, CDC37 (cell division cycle 37), is responsible for delivering BRAF (B-Rapidly Accelerated Fibrosarcoma) to the Hsp90 (heat shock protein 90) complex, where it is then translocated to the RAS (protooncogene product p21) complex at the plasma membrane for RAS mediated dimerization and subsequent activation. We identify a bipartite interaction between CDC37 and BRAF and delimitate the essential structural elements of CDC37 involved in BRAF recognition. We find an extended and conserved CDC37 motif, 20HPNID---SL--W31, responsible for recognizing the C-lobe of BRAF kinase domain, while the c-terminal domain of CDC37 is responsible for the second of the bipartite interaction with BRAF. We show that dimerization of BRAF, independent of nucleotide binding, can act as a potent signal that prevents CDC37 recognition and discuss the implications of mutations in BRAF and the consequences on signaling in a clinical setting, particularly for class 2 BRAF mutations.

Non-equilibrium protein folding and activation by ATP-driven chaperones

“…Both the N-terminal domain (NTD) and the C-terminal domain (CTD) of Cdc37 bind to the partially unfolded kinase [49, 50]. Individually NTD and CTD bind to the kinase with low affinities [50] (on the order of 100 µM), but the bipartite interaction between the complete Cdc37 and the kinase results in sub-micromolar affinity.…”

“…Both the N-terminal domain (NTD) and the C-terminal domain (CTD) of Cdc37 bind to the partially unfolded kinase [49, 50]. Individually NTD and CTD bind to the kinase with low affinities [50] (on the order of 100 µM), but the bipartite interaction between the complete Cdc37 and the kinase results in sub-micromolar affinity. Based on the cryo-EM structure of the Hsp90-kinase-Cdc37 complex, the bipartite interaction may lead to the encirclement of a Hsp90 protomer by the kinase-Cdc37 binary complex, thus preventing the kinase from slipping off Hsp90 (Fig.…”

“…A puzzling observation is that Cdc37 binds to both the inactive B-Raf kinase and the active B-Raf mutant B-Raf V600E (which has the valine at position 600 mutated to a glutamate) with similar affinities [51]: K D = 1.0 µM for the wild-type B-Raf and K D = 0.4 µM for the mutant B-Raf V600E [50]. This can be explained by the above proposal that Cdc37 binds with high affinity to the inactive-tending conformation of the kinase but with comparatively negligible affinity to the other conformations.…”

BRAF Δβ3-αC in-frame deletion mutants differ in their dimerization propensity, HSP90 dependence, and druggability

“…3, A, B, and D). On the basis of very recent insights from cryo-EM and deuterium exchange/MS experiments showing that the HSP90/CDC37 complex recognizes RAF molecules with N and C loop unfolded ( 101 – 103 ) and the notion that R-spine formation–induced conformational changes are a prerequisite for efficient N- and C-loop compaction and hence DIF-mediated dimerization ( 15 , 17 , 93 ), we posit that it is the high dimerization propensity of BRAF ∆LNVTAP>F that precludes its interaction with the chaperone complex. This hypothesis is supported by our experiment in which the R509H and AAE mutations increased HSP90 binding of BRAF ∆LNVTAP>F (Fig.…”

An Editorial on the Special Issue ‘Hsp90 Structure, Mechanism and Disease’

“…A second contribution by Bjorklund et al [ 18 ] looks at the activation of the class 2 BRAF mutant L597R. The authors discuss how BRAF addiction for Hsp90 is broken upon dimerization of the kinase.…”