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
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.
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- "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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- "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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- "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 . Protein samples were separated by SDS-PAGE gel, in-gel digested and analyzed by LC-MS and LC-MS/MS as described previously . "
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