HSP90-CDC37-PP5 forms a structural platform for kinase dephosphorylation

Abstract

Activation of client protein kinases by the HSP90 molecular chaperone system is affected by phosphorylation at multiple sites on HSP90, the kinase-specific co-chaperone CDC37, and the kinase client itself. Removal of regulatory phosphorylation from client kinases and their release from the HSP90-CDC37 system depends on the Ser/Thr phosphatase PP5, which associates with HSP90 via its N-terminal TPR domain. Here, we present the cryoEM structure of the oncogenic protein kinase client BRAFV600E bound to HSP90-CDC37, showing how the V600E mutation favours BRAF association with HSP90-CDC37. Structures of HSP90-CDC37-BRAFV600E complexes with PP5 in autoinhibited and activated conformations, together with proteomic analysis of its phosphatase activity on BRAFV600E and CRAF, reveal how PP5 is activated by recruitment to HSP90 complexes. PP5 comprehensively dephosphorylates client proteins, removing interaction sites for regulatory partners such as 14-3-3 proteins and thus performing a ‘factory reset’ of the kinase prior to release.

Structural insight into guanylyl cyclase receptor hijacking of the kinase–Hsp90 regulatory mechanism

“…The core structural principles of Cdc37 mediated client recruitment to Hsp90 appear to remain constant across its large range of client diversity. Across other client–Hsp90–Cdc37 complexes with canonical soluble kinase clients (Cdk4, RAF1, B-raf) (García-Alonso et al, 2022; Oberoi et al, 2022; Verba et al, 2016), we see a conserved role for Cdc37 in client recruitment by associating with the C-lobe at the N-, C-lobe interface (Supplemental Figure 2A). In these complexes we see high levels of structural conservation for the Hsp90–Cdc37 (Cα RMSDs of 1.4-3.3 Å for Hsp90 and 1.5-2.5 Å for Cdc37), while the client is structurally most homogenous at the interface with Cdc37, though less structurally conserved overall (Cα RMSDs of 3.5-11.6 Å).…”

“…The Cricetulus griseus HSP90β and Cdc37 show remarkable sequence conservation in comparison to the human equivalents, at 99.7 and 94.2% identity, respectively. This native pulldown strategy contrasts with the structures of Hsp90–Cdc37 in complex with soluble kinases (García-Alonso et al, 2022; Oberoi et al, 2022; Verba et al, 2016), for which Hsp90 and Cdc37 had to be overexpressed to obtain complex suitable for imaging. Three-dimensional reconstruction of our GC-C–Hsp90–Cdc37 particles generated a 3.9 Å resolution map of the regulatory complex (Figure 1, Supplementary Figure 1).…”

“…The present cryoEM structure of GC-C–Hsp90–Cdc37 resolves the loading of GC-C, via its PK domain and interaction with Cdc37, to the Hsp90 core dimer (Figure 1, 2). This complex shows significant structural similarity to the mechanism used to regulate soluble active kinases (García-Alonso et al, 2022; Oberoi et al, 2022; Verba et al, 2016) and presumably membrane receptor kinases in the human proteome. This structural and mechanistic conservation is largely driven by the co-chaperone Cdc37, which serves as the main binding platform for these clients by associating to the fold of the kinase (or pseudokinase in the case of mGC) domain, relatively independent of sequence identity.…”

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

“…Two recent studies might provide additional explanations for the contrasting HSP90 binding behavior of BRAF Δβ3-αC mutants (100,101). Using cryo-electron microscopy (cryo-EM), it was shown that the HSP90/CDC37 complex binds to the C-lobe of the kinase domains of BRAF and RAF1, while the latter, which shows higher affinity to the chaperone complex, also binds to the N-lobe and in the vicinity of the Δβ3-αC segment (101).…”

A Fluorescence-Based Sensor for Calibrated Measurement of Protein Kinase Stability in Live Cells

“…If the HSP90 ATPase activity is inhibited pharmacologically, HSP90 cannot load new clients through CDC37 and kinases that are normally HSP90 clients are instead subjected to ubiquitin-mediated degradation 11 . Structures of HSP90 and CDC37 in complex with cyclin-dependent kinase 4 28 , CRAF 29,30 , and BRAF(V600E) 31 revealed that the chaperone complex binds to a partially unfolded conformation of the kinase domain. Like other chaperones, it is likely that this interaction prevents further unfolding and aggregation of the client kinase domains that would be deleterious to the cell.…”