Substrate Binding Drives Large-Scale Conformational Changes in the Hsp90 Molecular Chaperone

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158-2517, USA.
Molecular cell (Impact Factor: 14.02). 04/2011; 42(1):96-105. DOI: 10.1016/j.molcel.2011.01.029
Source: PubMed


Hsp90 is a ubiquitous molecular chaperone. Previous structural analysis demonstrated that Hsp90 can adopt a large number of structurally distinct conformations; however, the functional role of this flexibility is not understood. Here we investigate the structural consequences of substrate binding with a model system in which Hsp90 interacts with a partially folded protein (Δ131Δ), a well-studied fragment of staphylococcal nuclease. SAXS measurements reveal that under apo conditions, Hsp90 partially closes around Δ131Δ, and in the presence of AMPPNP, Δ131Δ binds with increased affinity to Hsp90's fully closed state. FRET measurements show that Δ131Δ accelerates the nucleotide-driven open/closed transition and stimulates ATP hydrolysis by Hsp90. NMR measurements reveal that Hsp90 binds to a specific, highly structured region of Δ131Δ. These results suggest that Hsp90 preferentially binds a locally structured region in a globally unfolded protein, and this binding drives functional changes in the chaperone by lowering a rate-limiting conformational barrier.

    • "In this conformation, known cochaperone binding sites remain free and thus simultaneous binding of client and cochaperones may occur. The Tau binding site partially overlaps with the binding sites for Cdk4, GR, and the model substrate D131D (Genest et al., 2013; Street et al., 2011; Vaughan et al., 2006). Addition of ATP to the preformed Hsp90- Tau complex modulates Hsp90 conformational dynamics and most likely breaks the symmetry within the Hsp90 dimer, as indicated by splitting of some NMR signals. "
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    ABSTRACT: Hsp90 chaperones receive much attention due to their role in cancer and other pathological conditions, and a tremendous effort of many laboratories has contributed in the past decades to considerable progress in the understanding of their functions. Hsp90 chaperones exist as dimers and, with the help of cochaperones, promote the folding of numerous client proteins. Although the original view of these interactions suggested that these dimeric complexes were symmetrical, it is now clear that many features are asymmetrical. In this review we discuss several recent advances that highlight how asymmetric interactions with cochaperones as well as asymmetric posttranslational modifications provide mechanisms to regulate client interactions and the progression through Hsp90's chaperone cycle. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Apr 2015 · Molecular cell
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    • "To measure the re-opening rate, inter FRET-labeled hTRAP1 was pre-closed with AMPPNP. After closure was complete a 20-fold excess ADP was added such that upon re-opening of the NTD dimer interface, ADP would exchange resulting in a decreased FRET signal (Street et al., 2011). Previous studies found apo state nucleotide on and off-rates to be fast (Leskovar et al., 2008), thus the above experiment provides a good approximation of the uni-molecular reopening rate. "
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    ABSTRACT: Hsp90 is a conserved chaperone that facilitates protein homeostasis. Our crystal structure of the mitochondrial Hsp90, TRAP1, revealed an extension of the N-terminal β-strand previously shown to cross between protomers in the closed state. Here we address the regulatory function of this extension or 'strap' and demonstrate it's responsibility for an unusual temperature dependence in ATPase rates. This dependence is a consequence of a thermally-sensitive kinetic barrier between the apo 'open' and ATP-bound 'closed' conformations. The strap stabilizes the closed state through trans-protomer interactions. Displacement of cis-protomer contacts from the apo state is rate-limiting for closure and ATP hydrolysis. Strap release is coupled to rotation of the N-terminal domain and dynamics of the nucleotide binding pocket lid. The strap is conserved in higher eukaryotes but absent from yeast and prokaryotes suggesting its role as a thermal and kinetic regulator, adapting Hsp90s to the demands of unique cellular and organismal environments.
    Full-text · Article · Dec 2014 · eLife Sciences
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    • "To minimize the impact of the cross-linker on Hsp90 conformation, we preclosed the chaperone with AMPPNP. Previous work demonstrated that Δ131Δ can bind Hsp90 Ec in the closed state [18]. Protein samples were separated by SDS-PAGE gel, in-gel digested and analyzed by LC-MS and LC-MS/MS as described previously [35]. "
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    ABSTRACT: Hsp90 is a conformationally dynamic molecular chaperone known to promote the folding and activation of a broad array of protein substrates ("clients"). Hsp90 is believed to preferentially interact with partially folded substrates, and it has been hypothesized that the chaperone can significantly alter substrate structure as a mechanism to alter the substrate functional state. However, critically testing the mechanism of substrate recognition and remodeling by Hsp90 has been challenging. Using a partially folded protein as a model system, we find that the bacterial Hsp90 adapts its conformation to the substrate, forming a binding site that spans the middle and C-terminal domains of the chaperone. Crosslinking and NMR measurements indicate that Hsp90 binds to a large partially-folded region of the substrate and significantly alters both its local and long-range structure. These findings implicate Hsp90's conformational dynamics in its ability to bind and remodel partially folded proteins. Moreover, native-state hydrogen exchange indicates that Hsp90 can also interact with partially folded states only transiently populated from within a thermodynamically stable native state ensemble. These results suggest a general mechanism by which Hsp90 can recognize and remodel native proteins by binding and remodeling partially folded states that are transiently sampled from within the native ensemble.
    Full-text · Article · Apr 2014 · Journal of Molecular Biology
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