Dissection of the ATP-induced conformational cycle of the molecular chaperone Hsp90.
ABSTRACT The molecular chaperone heat-shock protein 90 (Hsp90) couples ATP hydrolysis to conformational changes driving a reaction cycle that is required for substrate activation. Recent structural analysis provided snapshots of the open and closed states of Hsp90, which mark the starting and end points of these changes. Using fluorescence resonance energy transfer (FRET), we dissected the cycle kinetically and identified the intermediates on the pathway. The conformational transitions are orders of magnitude slower than the ATP-hydrolysis step and thus are the limiting events during the reaction cycle. Furthermore, these structural changes can be tightly regulated by cochaperones, being completely inhibited by Sti1 or accelerated by Aha1. In fact, even in the absence of nucleotide, Aha1 induces Hsp90 rearrangements that speed up the conformational cycle. This comprehensive reconstitution of the Hsp90 cycle defines a controlled progression through distinct intermediates that can be modulated by conformation-sensitive cochaperones.
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ABSTRACT: The ubiquitous Hsp90 is critical for protein homeostasis in the cells, stabilizing "client" proteins in a functional state. Hsp90 activity depends on its ability to bind and hydrolyze ATP, involving various conformational changes that are regulated by co-chaperones, posttranslational modifications and small molecules. Compounds like geldanamycin (GA) and radicicol inhibit the Hsp90 ATPase activity by occupying the ATP binding site, which can lead client protein to degradation and also inhibit cell growth and differentiation in protozoan parasites. Our goal was to produce the recombinant Hsp90 of Leishmania braziliensis (LbHsp90) and construct of its N-terminal (LbHsp90N) and N-domain and middle-domain (LbHsp90NM), which lacks the C-terminal dimerization domain, in order to understand how Hsp90 works in protozoa. The recombinant proteins were produced folded as attested by spectroscopy experiments. Hydrodynamic experiments revealed that LbHsp90N and LbHsp90NM behaved as elongated monomers while LbHsp90 is an elongated dimer. All proteins prevented the in vitro citrate synthase and malate dehydrogenase aggregation, attesting that they have chaperone activity, and interacted with adenosine ligands with similar dissociation constants. The LbHsp90 has low ATPase activity (k(cat)=0.320min(-1)) in agreement with Hsp90 orthologs, whereas the LbHsp90NM has negligible activity, suggesting the importance of the dimeric protein for this activity. The GA interacts with LbHsp90 and with its domain constructions with different affinities and also inhibits the LbHsp90 ATPase activity with an IC(50) of 0.7μM. All these results shed light on the LbHsp90 activity and are the first step to understanding the Hsp90 molecular chaperone system in L. braziliensis.Biochimica et Biophysica Acta 08/2012; 834:351. · 4.66 Impact Factor
Article: Heat shock protein 90 from Escherichia coli collaborates with the DnaK chaperone system in client protein remodeling.[show abstract] [hide abstract]
ABSTRACT: Molecular chaperones are proteins that assist the folding, unfolding, and remodeling of other proteins. In eukaryotes, heat shock protein 90 (Hsp90) proteins are essential ATP-dependent molecular chaperones that remodel and activate hundreds of client proteins with the assistance of cochaperones. In Escherichia coli, the activity of the Hsp90 homolog, HtpG, has remained elusive. To explore the mechanism of action of E. coli Hsp90, we used in vitro protein reactivation assays. We found that E. coli Hsp90 promotes reactivation of heat-inactivated luciferase in a reaction that requires the prokaryotic Hsp70 chaperone system, known as the DnaK system. An Hsp90 ATPase inhibitor, geldanamycin, inhibits luciferase reactivation demonstrating the importance of the ATP-dependent chaperone activity of E. coli Hsp90 during client protein remodeling. Reactivation also depends upon the ATP-dependent chaperone activity of the DnaK system. Our results suggest that the DnaK system acts first on the client protein, and then E. coli Hsp90 and the DnaK system collaborate synergistically to complete remodeling of the client protein. Results indicate that E. coli Hsp90 and DnaK interact in vivo and in vitro, providing additional evidence to suggest that E. coli Hsp90 and the DnaK system function together.Proceedings of the National Academy of Sciences 05/2011; 108(20):8206-11. · 9.68 Impact Factor
Article: Regulation of Molecular Chaperones through Post-Translational Modifications: Decrypting the Chaperone Code.[show abstract] [hide abstract]
ABSTRACT: Molecular chaperones and their associated cofactors form a group of highly specialized proteins that orchestrate the folding and unfolding of other proteins and the assembly and disassembly of protein complexes. Chaperones are found in all cell types and organisms, and their activity must be tightly regulated to maintain normal cell function. Indeed, deregulation of protein folding and protein complex assembly is the cause of various human diseases. Here, we present the results of an extensive review of the literature revealing that the post-translational modification (PTM) of chaperones has been selected during evolution as an efficient mean to regulate the activity and specificity of these key proteins. Because the addition and reciprocal removal of chemical groups can be triggered very rapidly, this mechanism provides an efficient switch to precisely regulate the activity of chaperones on specific substrates. The large number of PTMs detected in chaperones suggests that a combinatory code is at play to regulate function, activity, localization, and substrate specificity for this group of biologically important proteins. This review surveys the core information currently available as a starting point towards the more ambitious endeavor of deciphering the "chaperone code".Biochimica et Biophysica Acta 02/2013; · 4.66 Impact Factor