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Elucidating the mechanism of substrate recognition by the bacterial Hsp90 molecular chaperone.

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. Cross-linking 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.

SUBMITTER: Street TO

PROVIDER: S-EPMC5322795 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Elucidating the mechanism of substrate recognition by the bacterial Hsp90 molecular chaperone.

Street Timothy O TO Zeng Xiaohui X Pellarin Riccardo R Bonomi Massimiliano M Sali Andrej A Kelly Mark J S MJ Chu Feixia F Agard David A DA

Journal of molecular biology 20140412 12

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 ...[more]

PMID: 24726919

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Project description:BackgroundPeptidyl-prolyl isomerases (PPIases) catalyze cis/trans isomerization of peptidyl-prolyl bonds, which is often rate-limiting for protein folding. SlyD is a two-domain enzyme containing both a PPIase FK506-binding protein (FKBP) domain and an insert-in-flap (IF) chaperone domain. To date, the interactions of these domains with unfolded proteins have remained rather obscure, with structural information on binding to the FKBP domain being limited to complexes involving various inhibitor compounds or a chemically modified tetrapeptide.ResultsWe have characterized the binding of 15-residue-long unmodified peptides to SlyD from Thermus thermophilus (TtSlyD) in terms of binding thermodynamics and enzyme kinetics through the use of isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, and site-directed mutagenesis. We show that the affinities and enzymatic activity of TtSlyD towards these peptides are much higher than for the chemically modified tetrapeptides that are typically used for activity measurements on FKBPs. In addition, we present a series of crystal structures of TtSlyD with the inhibitor FK506 bound to the FKBP domain, and with 15-residue-long peptides bound to either one or both domains, which reveals that substrates bind in a highly adaptable fashion to the IF domain through β-strand augmentation, and can bind to the FKBP domain as both types VIa1 and VIb-like cis-proline β-turns. Our results furthermore provide important clues to the catalytic mechanism and support the notion of inter-domain cross talk.ConclusionsWe found that 15-residue-long unmodified peptides can serve as better substrate mimics for the IF and FKBP domains than chemically modified tetrapeptides. We furthermore show how such peptides are recognized by each of these domains in TtSlyD, and propose a novel general model for the catalytic mechanism of FKBPs that involves C-terminal rotation around the peptidyl-prolyl bond mediated by stabilization of the twisted transition state in the hydrophobic binding site.

Dataset Information

Elucidating the mechanism of substrate recognition by the bacterial Hsp90 molecular chaperone.

Publications

Elucidating the mechanism of substrate recognition by the bacterial Hsp90 molecular chaperone.

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