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. 2006 Sep 1;23(5):697-707.
doi: 10.1016/j.molcel.2006.07.016.

Structure of an Hsp90-Cdc37-Cdk4 complex

Cara K Vaughan  1 , Ulrich Gohlke, Frank Sobott, Valerie M Good, Maruf M U Ali, Chrisostomos Prodromou, Carol V Robinson, Helen R Saibil, Laurence H Pearl
Affiliations

Structure of an Hsp90-Cdc37-Cdk4 complex

Cara K Vaughan et al. Mol Cell. .

Abstract

Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle.

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Figures

Figure 1
Figure 1. Purification and Composition of the Hsp90-Cdc37-Cdk4 Ternary Complex
a) 10% SDS-PAGE of purified Hsp90-Cdc37-Cdk4 (H-C-K) complex. b) Integrated Optical Density for components of the H-C-K complex for four successive two-fold dilutions in a Coomassie-stained 10% SDS-PAGE. The IOD was normalized by calculated molecular weight of each component and further normalized against the smallest component, Cdk4. The ratio of Cdc37 to Cdk4 remains 1:1 over successive dilutions, in agreement with results from mass spectrometry (see Figure 1d). The Coomassie-binding of Hsp90 is non-linear, possibly as a consequence of the protein’s very acidic nature. c) The final purification step is analytical gel filtration and the chromatogram shows that the proteins run as a single species. d & e) The Nano-ESI TOF MS spectrum of the complex shows the highest molecular weight species observed in the gas phase is a complex comprising a dimer of Hsp90, a monomer of Cdc37 and a monomer of Cdk4. Masses calculated from the sequences are: Hsp90, 82573 Da; Cdc37, 44468 Da; Cdk4, 35712 Da.
Figure 2
Figure 2. Purification and Composition of the Cdc37-Cdk4 Binary Complex
a) 10% SDS-PAGE of purified Cdc37-Cdk4 (C-K) complex. b) The final purification step is analytical gel filtration and the chromatogram shows that the proteins run as a single species. c & d) The Nano-ESI TOF MS spectrum of the complex shows the highest molecular weight species observed in the gas phase is a complex comprising a dimer of Cdc37 and a monomer of Cdk4. Masses calculated from the sequences are: Cdc37, 44468 Da; Cdk4, 35712 Da.
Figure 3
Figure 3. Negative-stain Electron Microscopy of Hsp90-Cdc37-Cdk4 Complex
a) A typical micrograph of 2% uranyl acetate-stained H-C-K showing views of the complex. Only particles that were well separated from the others and well stained were picked for analysis. Examples are highlighted in red. b) Reprojections from the model show features that are recognizable in typical raw particles. Some masked, centred raw particles retrospectively aligned to class averages generated by classification after the final alignment by projection matching. The corresponding reprojections of the model are shown below the class averages.
Figure 4
Figure 4. Three-dimensional Reconstruction of Hsp90-Cdc37-Cdk4 Complex
Three-dimensional reconstruction of the H-C-K complex from negative stain electron microscopy. a) Side views (related by 180º rotation around the vertical) - the oval channel running through the centre of the complex is immediately apparent, as are the two twisting columns of density connecting the base to the tip. b) End views showing the tip (right) and base (left). c) Secondary-structure cartoon (left) and molecular surface (right) of the Hsp90 dimer crystal structure (Ali et al., 2006). Models were generated using MacPyMOL (www.pymol.org).
Figure 5
Figure 5. Architecture of the Hsp90-Cdc37-Cdk4 Complex
a & b) Different views of the EM reconstruction, rotated around the vertical axis and corresponding views of the crystal structure, filtered to 20 Å. Equivalent monomers in the crystal structure and the EM reconstruction are outlined in black for each view. By comparison of the two, domains of Hsp90 can be identified. Where one domain is hidden behind another the outline is coloured yellow. There are some interdomain movements on complexation with Cdc37 and Cdk4. c) The absolute hand of the EM reconstruction can be assigned by matching the twist of the two structures. The C-terminal domain is easily recognised, therefore the polarity of Hsp90 within the model can be determined.
Figure 6
Figure 6. Modelling Cdc37 and Cdk4
a) The relative orientation of domains of the crystal structure of Hsp90 are adjusted by small rotations in the hinge regions and fitted into the EM reconstruction. The location of M-Cdc37 is determined by a least squares fit of the N-terminal domain of the open monomer with N-Hsp90 from the crystal structure of the complex with Cdc37. Cdk6 is used as a model for Cdk4. The larger C-terminal lobe of the kinase fills the larger lobe of the reconstruction and consequently is intimately associated with the middle-domain of one Hsp90 monomer. The N-terminal lobe is smaller and fills the remaining density, associating with either the N-terminal domain of Hsp90 or Cdc37, or both. Atomic models are shown superimposed on their molecular volumes filtered to 20Å resolution. The EM reconstruction is coloured according to protein chain: Hsp90 open monomer, orange; Hsp90 closed monomer, blue; Cdc37, green; Cdk4, red. b) Orthogonal views of the pseudo-atomic model of the (Hsp90)2-Cdc37-Cdk4 complex docked into the EM reconstruction. Some unassigned density in the vicinity of the Hsp90 N-termini (dotted outline) could accommodate the N-terminal domain of Cdc37, whose structure is not known, but is predicted to contain a segment of coiled-coil, and could bridge to the N-terminal domain of the closed Hsp90 monomer and/or the bound kinase.
Figure 7
Figure 7. Client Protein Interactions
Comparison of the ~19Å single-particle EM reconstruction (left) of an (Hsp90)2-Cdc37-Cdk4 complex with the ATP-bound Hsp90 crystal structure (right). The bilobal kinase client (red) appears to interact with the N-domain of one Hsp90 protomer and a region on the middle segment close to Trp300 (red on left), which is implicated in client protein binding. Changes in relative position of these Hsp90 domains coupled to the ATPase cycle would be transmitted to the bound client protein.
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