The HSP90-CDC37 Chaperone System Orchestrates RAF1 Kinase Activation Through a Pre-Dimerization Mechanism

Aizpurua, G.; Mesa, P.; de la Puente, L.; Ciges-Tomas, J. R.; Lomba, L.; Lechuga, C.; Zarzuela, E.; Isasa, M.; Van der Hoeven, L. R.; Olsen, J. V.; Barbacid, M.; Garcia-Alonso, S.; Montoya, G.

doi:10.64898/2026.03.25.713956

bioRxiv · 10.64898/2026.03.25.713956

The HSP90-CDC37 Chaperone System Orchestrates RAF1 Kinase Activation Through a Pre-Dimerization Mechanism

Aizpurua, G.·Mesa, P.·de la Puente, L.·Ciges-Tomas, J. R.·Lomba, L.·Lechuga, C.·Zarzuela, E.·Isasa, M.·Van der Hoeven, L. R.·Olsen, J. V.·Barbacid, M.·Garcia-Alonso, S.·Montoya, G.

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Abstract

RAF kinases activate MEK in the RAS-MAPK signaling pathway, and changes in RAF kinase signaling have been linked to tumor formation. RAF1 requires the HSP90-CDC37 chaperone system for proper activation, but how the HSP90-CDC37 chaperone system regulates RAF kinase maturation remains enigmatic. We present novel cryo-EM structures of previously uncharacterized RAF1 chaperone complexes, including a 2:2:2 RAF1-HSP90-CDC37 complex (RRHCC), intermediate assemblies (RHCC), and a RAF1-HSP90-CDC37-p23 complex (RHCp23). These reveal an asymmetric stepwise folding mechanism unique among HSP90 kinase clients in which one RAF1 threads through the HSP90 lumen while another is captured in a "casting mold" formed by CDC37 and HSP90 that stabilizes the partially folded C helix of RAF1. The RHCp23 structure shows how p23 cooperates with CDC37 to regulate ATP hydrolysis and client release. The HSP90-CDC37 system supports pre-dimerization of RAF1 and BRAFV600E homodimers and RAF1 heterodimers, a mechanism unique to RAF among kinase clients of HSP90. Phosphoproteomics reveals selective activating phosphorylations within RRHCC. These RAF isoform complexes differentially activate MEK signaling and cell proliferation, establishing HSP90-CDC37 as not just a passive stabilizer but an active regulator of RAF signaling with therapeutic implications.

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Aizpurua, G., Mesa, P., de la Puente, L., Ciges-Tomas, J. R., Lomba, L., Lechuga, C., Zarzuela, E., Isasa, M., Van der Hoeven, L. R., Olsen, J. V., Barbacid, M., Garcia-Alonso, S., Montoya, G.. 2026-03-27. The HSP90-CDC37 Chaperone System Orchestrates RAF1 Kinase Activation Through a Pre-Dimerization Mechanism. https://doi.org/10.64898/2026.03.25.713956

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