Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase

Abstract

The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" kinases and the reason why some kinases are addicted to Hsp90 while closely related family members are independent are unknown. Our structural understanding of these interactions is lacking, as no full-length structures of human Hsp90, Cdc37, or either of these proteins with a kinase have been elucidated. Here we report a 3.9 angstrom cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex. Surprisingly, the two lobes of Cdk4 are completely separated with the β4-β5 sheet unfolded. Cdc37 mimics part of the kinase N lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase β5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. On the basis of this structure and an extensive amount of previously collected data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions.

Fig. 1. The 4Å map of Hsp90/Cdc37/Cdk4

Density map colored by resolution. (B) hHsp90β model built into the density map, with different monomers colored shades of orange. Inserts show high resolution features. Arrows show density un-accounted for by Hsp90.

Fig. 2. Cdk4 is unfolded when complexed with Hsp90 and Cdc37

(A) Cdk4 (K4) C-lobe (blue) fit into the map (B) The tubular density from high-resolution map through the lumen of Hsp90 is perfectly fit by an unfolded β5 sheet (in sticks) of Cdk4. In magenta are previously disordered client interacting loops on Hsp90. (C) Cdk4/Hsp90 interface with hydrophobic residues in spheres, salt bridges in sticks.

Fig. 3. Rounds of focused 3D classification yield distinct densities for Cdc37 and the Cdk4 N-lobe

One of the new classes has clear density for the Cdc37 (37) M/C fragment crystal structure. In teal is our complete Cdc37 model (residues 1–260). Note the β-strand wrapping around the outside of Hsp90 connecting the two major Cdc37 domains. (B) Two additional classes show new density for the missing Cdk4 N-lobe. The classes minus the Hsp90/Cdc37 density, in blue and maroon, highlight the two new Cdk4 N lobe conformations. Fitted kinase models are in ribbons.

Fig. 4. Hsp90, Cdc37, and Cdk4 are intricately interwoven in the complex

Two views of the complete model, showing ribbon in (A) and surface in (B)

Fig. 5. High-resolution details of Cdc37 interactions with Hsp90 and Cdk4

Overall arrangement of Hsp90/Cdc37/Cdk4 (one Hsp90 monomer removed for clarity). The insets highlight Cdc37/Cdk4 interaction features: top – Cdc37/Cdk4 interact via hydrophobic interactions and backbone hydrogen bonds, with perfect shape complementarity, bottom - overlay of Cdc37’s conserved HPN motif (teal) perfectly mimicking type I β-turn of αC-β4 loop of 6 different kinases (shades of gray). (PDB codes: 3g33, 2itp, 1qmz, 3pp0, 1jnk, 4fk3) (B) Zooming in on Hsp90/Cdc37 interactions. Top insert: Cdc37/Hsp90 interactions mimic p23/Hsp90 interactions identified previously (magenta). Middle insert: phospho-Ser13 stabilizes local Cdc37 structure through interactions with conserved R36 and H33 and also interacts with Hsp90 at K406. Bottom insert: six salt bridges stabilizing Hsp90/Cdc37 interactions.

Fig. 6. Conceptual model for linkage between kinase folding and activation and proposed model for the Hsp90:Cdc37:kinase cycle

(A) Transitioning between states through an unfolded intermediate (dashed line) has a lower energy barrier than through rigid body motion (solid line). The Hsp90:Cdc37 stabilizes such an unfolded intermediate (orange solid line) (B). By comparison with non-clients (solid line), the active and inactive states of client kinases (dashed line) are de-stabilized. (C) Speculative model for an Hsp90:Cdc37:Kinase cycle. (I) The kinase domain transiently samples an open state. Interactions with co-factors (like cyclins, SH2/SH3 domains, etc.) stabilizes the kinase native state, disfavoring the open state. (NL-N-lobe, CYC-Cyclin) CKII phosphorylated Cdc37 captures the open state by binding the kinase C-lobe (II). Cdc37/kinase then binds to open Hsp90 (III). Hsp90 binds to ATP and closes upon the unfolded part of the kinase. Cdc37 migrates down, resulting in the structure described here (IV). Upon hydrolysis of ATP, Hsp90 opens with Cdc37/Cdk4 still bound, giving a chance for the kinase to fold (V). If it folds, it displaces Cdc37 and leaves the complex. If however it fails to displace Cdc37, then Hsp90 is able to re-bind ATP and go back to state IV, repeating the process. At some point during this cycle, PP5 phosphatase is recruited to the complex to de-phosphorylate Cdc37.