ATP-induced protein Hsp70 complex dissociation requires K+ but not ATP hydrolysis

Abstract

The molecular chaperone proteins, particularly Hsp60 and Hsp70, have been implicated in essential cell functions under both normal and stress conditions (reviewed in refs 1-5). Members of the family of heat-shock proteins of M(r) 70K, Hsp70, bind to unfolded proteins and short peptides. Addition of Mg-ATP results in the dissociation of the substrate polypeptides from the chaperone, but as ATP-gamma S (an ATP analogue that is only slowly hydrolysable) cannot substitute for ATP in this reaction, it has been concluded that ATP hydrolysis is necessary to dissociate Hsp70-substrate protein complexes. By independently measuring the rates of ATP hydrolysis and substrate protein dissociation, we show here that Mg-ATP binding but not Mg-ATP hydrolysis is essential for substrate dissociation. We also show that there is an absolute requirement for K+ for the effect of Mg-ATP: only the combination of K+ and Mg-ATP will cause the conformational change in Hsp70 that is necessary for substrate dissociation. Moreover, in the absence of K+, Mg-ATP favours complex formation. We consider these results in terms of a G-protein-like model.

A 70-kDa molecular chaperone, DnaK, from the industrial bacterium Bacillus licheniformis: gene cloning, purification and molecular characterization of the recombinant protein

“…In vitro studies have shown that Hsp70/DnaK proteins bind both denatured proteins and some short peptides, and release these substrates in response to the addition of ATP [47,48]. Hsp70/DnaK proteins have a weak ATPase activity and a peptide binding activity with a mechanism of coupling these two activities such that the addition of ATP induces peptide release [5,49]. The open/closed state of the Hsp70/ DnaK substrate-binding domain, which is mediated by the repositioning of a α-helical lid over the substrate-binding pocket, is governed by the nucleotide occupancy and status in the ATPase domain [5,50].…”

Ubiquitin-dependent Degradation of Certain Protein Substrates in Vitro Requires the Molecular Chaperone Hsc70

“…One well known and carefully studied function of molecular chaperones involves refolding of denatured proteins to their native form. The process is gradual and involves repeated, cation-dependent cycles of associationdissociation (5,12,13). It is possible that Hsc70 is involved in an attempt to refold denatured proteolytic substrates.…”

“…1 and Table II). In a search for a potential role of the cations in the proteolytic process, we noted that several functions of the 70-kDa heat shock cognate protein (Hsc70), such as peptide binding and release and clathrin uncoating, require K ϩ (12,13,41). The cation is required, most probably, to allow the change in the conformation of the chaperone that is necessary for substrate dissociation.…”

“…The ATPase activity in the chaperone hydrolyzes the Hsc70-bound ATP to ADP, which results in rebinding of the released substrate (12). The dissociation of the substrate is cation-dependent (12). K ϩ , for example, causes the conformational change in Hsc70 that is necessary for substrate dissociation; in the absence of the cation, MgATP favors the stable complex conformation.…”

“…Exchange of ADP with ATP lowers the affinity and results in the release of the substrate. The ATPase activity in the chaperone hydrolyzes the Hsc70-bound ATP to ADP, which results in rebinding of the released substrate (12). The dissociation of the substrate is cation-dependent (12).…”