The COOH-terminal Peptide Binding Domain Is Essential for Self-association of the Molecular Chaperone HSC70

Abstract

We have previously shown that the molecular chaperone HSC70 self-associates in solution into dimers, trimers, and probably high order oligomers, according to a slow temperature-and concentration-dependent equilibrium that is shifted toward the monomer upon binding of ATP peptides or unfolded proteins. To determine the structural basis of HSC70 self-association, the oligomerization properties of the isolated amino-and carboxyl-terminal domains of this protein have been analyzed by gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Whereas the amino-terminal ATPase domain (residues 1-384) was found to be monomeric in solution even at high concentrations, the carboxyl-terminal peptide binding domain (residues 385-646) exists as a slow temperature-and concentration-dependent equilibrium involving monomers, dimers, and trimers. Members of the highly conserved 70-kDa heat shock protein family (HSP70) 1 are involved in several cellular processes such as protein folding, assembly and disassembly of multimeric proteins, protein translocation across membranes, protein degradation, and signal transduction (for reviews see Refs. 1-4). They are thought to act as molecular chaperones by transiently binding hydrophobic regions exposed to the solvent in the nonnative conformations of proteins, thereby preventing off-pathway reactions that lead to aggregation (5).A prominent member of this family, the mammalian, constitutively expressed, 70-kDa heat shock cognate protein (HSC70), has been shown to bind peptides and unfolded proteins (6 -8) and to possess refolding activity in the presence of the cochaperone DnaJ (9, 10). HSC70 shows a very weak ATPase activity that can be stimulated 2-5-fold upon binding of peptides, unfolded proteins, clathrin light chains, and cochaperones of the DnaJ family (11-14). HSC70 seems to function through cycles of binding and release of polypeptide substrates coupled to binding and hydrolysis of ATP (15), in a mechanism involving cochaperones of the DnaJ protein family and a newly isolated factor, Hip (16). HSC70 is made of two domains, an NH 2 -terminal domain of 44 kDa (residues 1-384), which binds and hydrolyzes ATP, and a COOH-terminal domain of about 30 kDa (residues 385-646), which contains the peptide binding site (17, 18). The threedimensional structure of the NH 2 -terminal ATPase domain has been solved by crystallography (19), and the secondary structure topology of the peptide binding site (residues 385-543) has been determined by NMR methods (20). Recently, the structure of a complex between a seven-residue peptide and the COOHterminal domain of DnaK, the bacterial HSP70, has been established (21). However, the three-dimensional structure of the entire protein is still unknown.Self-association is a general and well conserved feature of the HSP70 protein family. BiP (22-24), the constitutive HSC70 (6, 25-30), the heat shock-inducible HSP70 (31), plant HSP70 (32), and DnaK (33), all show self-association properties. Nevertheless, the structural basis an...

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

“…The 22-kDa species seemed to be a degraded product of the 24-kDa hHsp70 520-641. These findings confirmed the results of the previous study (19) demonstrating that the dimerization as well as the oligomerization of Hsp70 was mediated through the C-terminal client-binding domain. Moreover, the results in Fig.…”

“…Previous studies consistently showed that the C-terminal client-binding domain is responsible for the dimer/oligomer formation of Hsc70 (19). However, there was an apparent discrepancy as to the role of sub-domains, whether the client-binding sub-domain or the lid sub-domain is critical for this role.…”

“…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.…”

Visualization and Mechanism of Assembly of a Glucocorticoid Receptor·Hsp70 Complex That Is Primed for Subsequent Hsp90-dependent Opening of the Steroid Binding Cleft

“…Thus, it seems inappropriate at this time to form rigid models of the priming step based on stoichiometry data that require the action of specifically a monomer or a dimer of hsp70 on the receptor. It is possible that only one molecule of hsp70 is bound directly to and interacting productively with the receptor, and that under these conditions of assembly with purified proteins the receptor-bound hsp70 can associate with other molecules of hsp70 to form dimers and trimers, much as purified hsp70 and its homologs have been shown to do in solution (32)(33)(34). It is perhaps important to note that analysis of both the native and assembled hsp90⅐Hop⅐hsp70 machinery has revealed a molar ratio of 1 hsp70 per hsp90 dimer (6,18).…”

“…Bacterial DnaK and its mammalian mitochondrial and cytoplasmic homologs self-associate in solution into dimers, trimers, and probably higher oligomers, with the equilibrium between these forms being dependent upon the concentration of the chaperone and the equilibrium being shifted toward the monomer by ATP or binding of peptide substrate (32)(33)(34). Under concentrated conditions, our purified hsp70 is predominantly dimeric on native gel electrophoresis with some trimers being detectable, yet under dilute conditions in reticulocyte lysate, the free hsp70 behaves as a monomer on molecular sieve chromatography (6), as did our purified hsp70 (data not shown).…”