Hsp90:client interactions have proven difficult to study in-vitro likely because the chaperone favors interactions with partially folded or metastable client states that are only transiently populated. Here our aim has been to test the use of a model system of non-native states for probing Hsp90:substrate interactions. Our choice of system was guided by the fact that although globally unfolded, Δ131Δ has significant residual structure, is only marginally unstable, but is soluble and non-aggregating even at high concentrations. Using a combination of SAXS, FRET, fluorescence anisotropy and NMR, we have found that HtpG binds a specific region of Δ131Δ and this binding results in large-scale conformational and functional changes to the chaperone. These findings reveal basic steps in the Hsp90: Δ131Δ nucleotide cycle ().
Our SAXS measurements and modeling suggest that under apo conditions HtpG adapts its conformation to Δ131Δ by a partial closure (). This structural analysis was aided by localizing Δ131Δ with ab-initio
reconstructions prior to structure-based fitting and rigid-body analysis. The SAXS fitting on full-length HtpG shows that upon binding Δ131Δ the chaperone adopts an equilibrium between a ‘V’-shaped conformation and a fully open state (, Supplemental Table 1
), indicating that Hsp90 maintains significant flexibility even after substrate loading. The residual flexibility suggests that Hsp90 could accommodate other cochaperones or binding partners within the loaded conformation. Also, to advance through the nucleotide cycle, Hsp90 must undergo large conformational changes requiring significant structural plasticity to reach the ATP state. While our SAXS modeling suggests that Δ131Δ remains bound to roughly the same region on HtpG, it is likely that concomitant with HtpG closure, there is some alteration in Δ131Δ:HtpG interactions and perhaps in Δ131Δ structure.
The apo, substrate-bound conformation of Hsp90 has a significant impact on the kinetics of the nucleotide cycle. Following previous work (Hessling et al., 2009
; Mickler et al., 2009
), we used kinetic FRET measurements to show that closure to the ATP state is significantly accelerated by Δ131Δ (). This closure acceleration is paralleled by an ATPase acceleration (Supplemental Figure 5b
), similar to reports of ATPase stimulation of human Hsp90 by the ligand binding domain of the glucocorticoid receptor (McLaughlin et al., 2002
). Our findings suggest that closure is rate limiting in ATP hydrolysis by Hsp90, and that client binding activates the chaperone by lowering this rate-limiting conformational barrier. The coupling between client binding, Hsp90 conformational changes and subsequent ATP hydrolysis, suggests a simple mechanism by which Hsp90 restricts unnecessary ATP utilization while maximizing efficiency of client activation.
The ATP state transition involves numerous structural changes: (i) ATP binding restructures an N-terminal helical region that makes cross-monomer contacts, (ii) dramatically changes the NTD/MD orientation leading to an interaction between a highly conserved arginine (residue 336 in HtpG) and the ATP γ-phosphate, and (iii) is associated with the release of a β-strand that is swapped across monomers stabilizing N-terminal dimerization, however it is not known which of these processes (or others) are rate-limiting. Indeed, our SAXS measurements are too low resolution to conclude whether Δ131Δ contacts are restricted to a single monomer or whether Δ131Δ-induced closure is driven by cross monomer contacts. A detailed study is needed to address these points.
The results with Δ131Δ suggest that Hsp90’s conformational plasticity is functionally important and may allow it to adapt to structurally diverse substrates, which catalyze further structural changes that lead to ATP hydrolysis. This implies that Hsp90’s flexibility should be conserved, which is indeed true for the homologs investigated by SAXS (HtpG, Hsc82, hHsp90a, Grp94 and TRAP, (Krukenberg et al., 2009a
) and unpublished observations). For bacterial, yeast and human Hsp90, detailed electron microscopy measurements have shown that a three-state apo-ATP-ADP conformational cycle is conserved but that the equilibria between states is species-specific (Southworth and Agard, 2008
). This result suggested that the Hsp90 conformational equilibrium is tuned to the specific substrate/cochaperone requirements of each organism. Indeed, Δ131Δ binds to the bacterial, yeast, and human Hsp90 homologs and affects their conformations however the relative magnitude of these structural changes are species-specific (Supplemental Figure 1
The conformational diversity and large structure of Hsp90 suggests that it can provide a combinatorial set of binding surfaces and conformations for interacting with structurally diverse substrates. An electron microscopy reconstruction of an Hsp90-Cdc37-Cdk4 complex (Vaughan et al., 2006
) shows that Hsp90 adopts a more closed conformation in complex with the kinase-cochaperone complex than we observe with Δ131Δ. This observation suggests that different substrates and cochaperone complexes can be accommodated by different Hsp90 conformations and possibly have different nucleotide cycle dependences.
Our NMR measurements suggest that Hsp90 selectively interacts with a region of Δ131Δ (residues ~ 85–110) that has been shown to have significant structure despite the fact that Δ131Δ is globally unfolded (Alexandrescu et al., 1994
; Alexandrescu and Shortle, 1994
; Ohnishi and Shortle, 2003
; Wang and Shortle, 1995
). Hsp90 is often referred to as operating in later stages of client folding, consistent with this finding. Future studies will be required to reveal whether binding changes the structure within this region, which specific elements of Hsp90 are involved in binding, and the impact of nucleotide-induced conformational changes in Hsp90. Inspection of the fractional peak height distribution in also shows decreased peak heights towards the C-terminus of Δ131Δ, possibly indicating a second binding site. Clearly, although SAXS measurements are ideal for characterizing the flexible Hsp90 conformation and the influence of Δ131Δ, high-resolution measurements are needed to elucidate these molecular details.