Oligomerization of the 17‐kDa peptide‐binding domain of the molecular chaperone HSC70

Abstract

Crystallographic and biochemical studies have indicated that the peptide-binding site of the molecular chaperone HSC70 is located in a small subdomain comprising a b-sheet motif followed by a helical region, and there is some evidence of the involvement of this site in oligomerization of the protein.To determine the structure of this subdomain in solution and examine its involvement in oligomerization of HSC70, a 17-kDa protein (residues 385±540 of HSC70) consisting mainly of the peptide-binding site was constructed and analyzed for oligomerization properties. This small domain was found to bind peptides and to form oligomers in solution, probably tetramers, which dissociated into monomers on peptide binding in a manner comparable with that observed for the whole protein. Furthermore, in the 60-kDa fragment of HSC70, which is composed of the 17-kDa domain and the 44-kDa ATPase domain, not only were the oligomerization properties conserved, but dissociation of multimeric species into monomers on ATP binding also occurred and peptide stimulation of ATPase activity was restored. These results indicate that the isolated 17-kDa peptide-binding domain is necessary and sufficient for oligomerization of the whole protein, suggesting that the peptide-binding site may be involved in the oligomerization process.Keywords: analytical ultracentrifugation; heat shock proteins; HSC70; molecular chaperones; self-association.The constitutively expressed 70-kDa heat shock cognate protein (HSC70), a member of the highly conserved 70-kDa heat shock protein family (HSP70), plays an important role in protein folding, assembly and disassembly, transport and signal transduction (for review see [1±3]). HSC70 is composed of an Nterminal ATPase domain (residues 1±384) [4] and a C-terminal domain (residues 385±646) which can be divided into a peptidebinding subdomain (residues 385±540) and a C-terminal subdomain (residues 540±646) [5,6]. The three-dimensional structure of the isolated N-terminal ATPase domain of HSC70 [7] and the C-terminal domain of the bacterial HSP70, DnaK, complexed with peptide [6], have been solved by crystallography. However, structural information for the whole protein is still lacking. At the functional level, HSC70 binds peptides and unfolded proteins [8,9]. It has very weak ATPase activity which can be stimulated two-to fivefold on binding of peptides [10] Proteins of the HSP70 family from bacteria to man oligomerize into several species in vitro [19±21] as well as in vivo [22±24]. Although the role of such a property has not yet been established, mounting evidence suggests the existence of a relationship between oligomerization and chaperone activity. Indeed, like chaperone activity, oligomerization of the HSP70s is regulated by ATP binding, co-chaperones and peptides [25±27], and the C-terminal domain of the protein (residues 385±646), which is necessary for chaperone activity as it contains the peptide-binding site, has recently been implicated in the process [26].The C-terminal domain of HSC70 is composed of ...

A Disulfide Bridge Mediated by Cysteine 574 Is Formed in the Dimer of the 70-kDa Heat Shock Protein

“…The 10-kDa C-terminal lid sub-domain (amino acids 542-646) of rat Hsc70, which forms an elongated bundle-like structure, dictates the self-association (22). In contrast, Fouchaq et al (20) reported that the 17-kDa peptide-binding sub-domain (amino acids 385-540 of Hsc70) was involved in the oligomerization process. The results obtained in the present study do not accord with either of these reports.…”

“…There is some evidence that the 30-kDa C-terminal client-binding domain is responsible for the dimerization of 70-kDa heat shock cognate protein Hsc70 (19,20). It has been reported that the 30-kDa C-terminal domain can be divided into N-terminal 18-kDa and C-terminal 10-kDa sub-domains.…”

“…Fouchaq et al (20) reported that the 17-kDa client-binding sub-domain of Hsc70 could be oligomerized.…”

Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody

“…The TKD sequence, which is exposed to the extracellular milieu of tumors, resides in the C-terminally localized oligomerization domain of the Hsp70 molecule (11). Furthermore, this TKD peptide in combination with low-dose IL-2 has been found to stimulate the migratory and cytolytic activity of NK cells against membrane Hsp70 + tumor cells (12).…”

Characterization of a Lidless Form of the Molecular Chaperone DnaK

“…ATP binding to the Nterminal domain induces a global conformational change in DnaK (7) which is thought to displace the lid from the top of the ␤-sandwich subdomain, creating the low affinity form of the protein. To gain insight into the function of the lid, slightly different lidless forms of DnaK and eukaryotic Hsp70s have been engineered (9,(15)(16)(17)(18). What has emerged from these studies is that loss of the lid does not interfere with interdomain coupling (15), and loss of the lid results in a 2-20-fold increase in K d values for peptide interaction with the ADPbound state of lidless DnaK (17,18).…”