Targeted disruption of Hsp90-N–Sgt1-CS interaction. (A) Yeast two-hybrid analysis of AtSgt1a-CS mutants. AtSGT1a or its derivatives were assayed for interaction with AtRAR1 and HvHSP90-N using LexA Yeast Two-Hybrid system. Mutation of residues Tyr157, Phe168, Lys221 or Glu223, in the conserved interface with Hsp90-N, abrogated reporter activation. (B) Co-precipitation assay with His₆-tagged human Hsp90β, and human Sgt1. Hsp90 efficiently co-precipitates wild-type Sgt1, but not mutants of the core interface residue, Tyr145 (equivalent to Tyr157 in the plant protein). (C) Co-precipitation assay with GST-tagged Arabidopsis Sgt1a, and plant Hsp90. AtSgt1a co-precipitates wild-type TaHsp90 or TaHsp90 with mutation of a residue not in the observed interface, but does not efficiently co-precipitate TaHsp90 with mutations in interfacial residues Ser89 or Val92.

Functional dependence of Sgt1 on Hsp90 interaction in vivo. (A) Functional assay of SGT1a mutants in Rx-mediated resistance against potato virus X (PVX). Rx-containing N. benthamiana plants silenced for NbSGT1 were co-infiltrated with Agrobacterium expressing wild-type AtSGT1a (lower left, positive control), or AtSGT1a mutants as indicated (right half of the leaf) or GUS (upper left, negative control) together with PVX-GFP. Virus accumulation was monitored by GFP fluorescence under UV illumination 5 days after inoculation. Mutations in residues not involved in the core interface (Tyr199 and Thr220) with Hsp90 had little effect on the ability of AtSgt1a to facilitate viral resistance. Mutations of the hydrophobic Hsp90 interface residues Tyr157 or Phe168, and charge-reversal mutations in polar interface residues Lys221 or Glu223, severely impaired the biological function of Sgt1a. (B) Single-point mutations in Hsp90-interacting residues did not significantly impair essential Sgt1 functions in yeast (left), but double mutants abolished viability (right). (C) Single-point mutations in Hsp90-interacting residues in yeast Sgt1 sensitize yeast to killing by the Hsp90 inhibitor geldanamycin (GA).

CS domain interactions. (A) Comparison of the binding surfaces used by the structurally homologous CS domains of the Hsp90 co-chaperones Sgt1 (left) and p23/Sba1 (right). Sgt1-CS uses residues on the face of its four-stranded β-sheet, whereas p23/Sba1 interacts with Hsp90-N through a C-terminal extension not present in Sgt1. The interaction site on Hsp90-N (bottom, magenta) is also completely different, with Sgt1-CS binding the side of the domain, whereas p23/Sba1 binds directly to the lid segment in its closed ATP-bound conformation. (B) Hypothetical model of Sgt1-CS binding to Hsp90 in the closed ATP-bound conformation, made by superimposing the Hsp90-N domain from the present structure on to the crystal structure of the full-length Hsp90-AMPPNP-p23/Sba1 structure (PDB code 2CG9). Consistent with the preference of Sgt1 for binding to the open ADP-bound state of Hsp90, the docked Sgt1-CS domains clash sterically, and this is likely to be exacerbated in the context of the full-length protein by the TPR and SGS domains, which extend from the N- and C terminus, respectively.

Crystal structure of Hsp90-N-Sgt1-CS complex. (A) In the crystals, opposite faces of the Sgt1-CS domain β-sandwich structure interact with different parts of adjacent Hsp90-N domains. (B) Conservation colouring of the Sgt1-CS surface (blue → red; most → least conserved) based on the alignment in Supplementary Figure 2. The surface shown on the left is highly conserved with a ridge of essentially invariant residues that form the interface with Hsp90. The very high degree of conservation suggests that this interface is biologically authentic, whereas that on the right is a crystal lattice contact. (C) Detail of the conserved Hsp90-N–Sgt1-CS interface. The interface is built around two hydrophobic patches, centred on Sgt1-Tyr157 and Phe168, reinforced by a network of hydrogen bonding interactions (dotted lines). Formation of the complex buries ∼1100 Ų of molecular surface, which is consistent with a reversible interface. (D) Overview of the Hsp90-N–Sgt1-CS interaction based on the conserved interface. The ADP bound in the pocket of the Hsp90-N domain and the mobile ‘lid' segment that closes in the ATP-bound state of Hsp90 are indicated.

Sgt1 interactions with SCF complex. (A) Bridged co-precipitation assay with His₆-tagged human Hsp90β, Sgt1 and Skp1. Hsp90 efficiently co-precipitates Skp1 (visualized by western blot) only when wild-type Sgt1 is present to bridge the interaction. Mutations in the Sgt1 Hsp90-binding residue Tyr145 prevent Skp1 co-precipitation. (B) Direct co-precipitation of Skp1 by Sgt1 (visualized by western blot) is not affected by Sgt1 Tyr145 mutations. (C) Sgt1 is co-precipitated by GST–Skp1–Skp2 or GST–Skp1–Skp2(F-Box) complexes, with no competitive displacement of Skp2 by increasing concentrations of Sgt1. Input protein loadings (10%) are visualized with Coomassie Brilliant Blue (CBB) and co-precipitated proteins are visualized by western blot. (D) Co-precipitation of Skp1 by GST-tagged Sgt1 is diminished by increasing concentrations of Cul1, showing competition between Sgt1 and Cul1 for binding to Skp1. Protein is visualized as in (C). (E) His₆-tagged Cul1 efficiently co-precipitates Skp1, but not Sgt1, showing that there is no direct interaction between Cul1 and Sgt1, and that Skp1 cannot bind Cul1 and Sgt1 simultaneously. Increasing concentrations of Sgt1 fail to displace Cul1, which binds Skp1 ∼70-fold tighter than Sgt1.

Co-binding of TPR domain E3 ligase CHIP and Sgt1. His₆-tagged human Hsp90β efficiently co-precipitates Sgt1 and CHIP, confirming that the TPR domain of Sgt1 does not interact with the C-terminal MEEVD sequence of Hsp90, which provides the binding site for CHIP, and other TPR domain co-chaperones.

A role for Hsp90–Sgt1 in SCF assembly. (A) Previous observations and data presented here that Hsp90–Sgt1 can interact with Skp1 in complex with an F-box protein such as Skp2. In such a complex, a client/substrate protein (S) could be bound to either the chaperone or to the F-box protein, and in principle could be transferred from one to the other. (B) Competitive displacement of Hsp90–Sgt1 from Skp1 by Cul1–Rbx1, concomitant with the release of Cul1–Rbx1 from Cand1, would permit formation of an active SCF complex, able to be neddylated and bind E2. Disruption of the Cand1–Cul1 interaction might require the ATPase activity of Hsp90. (C) Alternatively, as a parallel pathway during stress conditions or when the SCF route is overloaded, an Hsp90–Sgt1–Skp1–F-box protein complex could allow ubiquitination of a client/substrate protein by the recruitment of CHIP to the C-terminal binding site on Hsp90.