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. 2006 Apr 20;440(7087):1013-7.
doi: 10.1038/nature04716.

Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex

Maruf M U Ali  1 , S Mark Roe, Cara K Vaughan, Phillipe Meyer, Barry Panaretou, Peter W Piper, Chrisostomos Prodromou, Laurence H Pearl
Affiliations

Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex

Maruf M U Ali et al. Nature. .

Abstract

Hsp90 (heat shock protein of 90 kDa) is a ubiquitous molecular chaperone responsible for the assembly and regulation of many eukaryotic signalling systems and is an emerging target for rational chemotherapy of many cancers. Although the structures of isolated domains of Hsp90 have been determined, the arrangement and ATP-dependent dynamics of these in the full Hsp90 dimer have been elusive and contentious. Here we present the crystal structure of full-length yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1. The structure reveals the complex architecture of the 'closed' state of the Hsp90 chaperone, the extensive interactions between domains and between protein chains, the detailed conformational changes in the amino-terminal domain that accompany ATP binding, and the structural basis for stabilization of the closed state by p23/Sba1. Contrary to expectations, the closed Hsp90 would not enclose its client proteins but provides a bipartite binding surface whose formation and disruption are coupled to the chaperone ATPase cycle.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Architecture of Hsp90-p23/Sba1 Complex
a) Backbone tracing of the (Hsp90)2-(p23/Sba1)2 complex; Hsp90 - blue and orange, p23/Sba1 - red and green. All molecular graphics were produced with MacPyMOL (www.pymol.org) b) Molecular surface equivalent of a). The left-handed twist of the Hsp90 molecules is evident. c) As b) the projecting β-ribbon is connected by the truncated charged-linker, which is disordered in the crystals. d) As c) but rotated by 90° around the horizontal so that the exchange of the N-terminal strands is evident. e) Orthogonal view of Hsp90 protomer, rainbow coloured from the N-terminus (blue) to the C-terminus (red).
Figure 2
Figure 2. ATP-Dependent Conformational Changes
a) Comparison of the dimerised M-C construct (left), and equivalent regions from the Hsp90-p23/Sba1 complex (right). ATP-dependent association of the N-domains brings the the middle segments ~20Å closer together. The central ‘thumbnail’ of the Hsp90 dimer shows the direction of view and location of the M-C region in the overall structure. b) Secondary structure cartoon of the strand-swapped interface between the N-domains of Hsp90, viewed from the base (left) and side (right). c) Comparison of the isolated N-domain (left), and the AMP-PNP-bound state (right). In addition to lid closure, there is considerable movement of the N-terminal helix and complete detachment of the N-terminal strand, which swaps into the other N-domain of the dimer (shown as a ghost).
Figure 3
Figure 3. Domain Interfaces and Active Site Formation
a) Interfaces between the Hsp90 N-domains and middle segments: the inter-molecular N-domain (pale blue background), inter-molecular N-domain – middle (pale pink), and intra-molecular N-domain – middle segment (pale yellow) that assembles the catalytic apparatus. b) Details of the split catalytic apparatus. The γ-phosphate of AMP-PNP is orientated for attack by a water activated by Glu33. Arg380 polarises the γ-β phosphodiester bond and neutralises the transition state. Mutation of Glu33 or Arg380 kills catalysis; mutation of Asp79 abolishes ATP binding , . The mesh shows Fo-Fc ‘omit’ electron-density for the catalytic loop (red) and ATP-analogue (orange).
Figure 4
Figure 4. Mechanism of ATPase Regulation by p23/Sba1
a) Overall view of one p23/Sba1 molecule (green) packed against the Hsp90 dimer (cyan and orange), and interacting with the ‘lid’ (yellow) in its ATP-dependent closed conformation. Details are shown in b) and c). b) Residues 113-118 of p23/Sba1 (green) pack onto the ‘lid’ (orange). Mutation of Ile117 causes severe Sba1 loss of function in yeast. c) The conserved DFxxW motif of p23/Sba1 binds across the inter-molecular inter-domain interface, reinforcing the activated conformation of the catalytic loop carrying Arg380. d) Binding sites for p23/Sba1 (left) and Aha1 (right) overlap, so that their recruitment to Hsp90 is mutually exclusive.
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References

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