Effect of Nucleotides, Peptides, and Unfolded Proteins on the Self-association of the Molecular Chaperone HSC70

Abstract

In a previous study, we showed that the molecular chaperone HSC70 self-associates in solution in a reversible and likely unlimited fashion. Here, we examine the influence of nucleotides, nucleotide analogs, peptides, and unfolded proteins on the self-association properties of this protein. Whereas in the presence of ADP, HSC70 exists as a slow, concentration-and temperaturedependent monomer-oligomer equilibrium, in the presence of ATP, the protein is essentially monomeric, indicating that ATP shifts this equilibrium toward the monomer by stabilizing the monomer. Dissociation of oligomers into monomers is also obtained with the slowly hydrolyzable ATP analogs, adenosine 5-O-(thiotriphosphate) and 5-adenylyl-␤,␥-imidodiphosphate, or the complex between ADP and the phosphate analog, BeF 3 , indicating that binding but not hydrolysis of ATP is necessary and sufficient for the stabilization of HSC70 monomer. Furthermore, binding of short peptides or permanently unfolded proteins to the peptide binding site of HSC70 promotes the dissociation of oligomers into monomers, suggesting that protein substrates are able to compete with HSC70 for the same binding site. Because the release of peptides or unfolded proteins from HSC70 has also been shown to require ATP binding, these results indicate that dissociation of oligomers is controlled by a mechanism similar to that of release of protein substrates and suggest that binding of HSC70 to itself occurs via the peptide binding site and mimics binding of HSC70 to protein substrates.The correct folding of proteins is thought to be the result of a kinetic competition between a productive "on-pathway" leading to the native, biologically active, state and an abortive "offpathway" leading to aggregation of incompletely folded intermediates (1-4). In vivo, the efficiency of the folding process depends on the presence of cellular factors such as molecular chaperones, which by transiently binding to unfolded or partially folded polypeptide chains prevent incorrect intra-and intermolecular interactions, thus favoring the productive pathway over aggregation (4 -6). Among these factors, the 70-kDa heat shock cognate protein (HSC70), a constitutively expressed member of the highly conserved 70-kDa heat shock protein (HSP70) family, plays an essential role not only in protein folding but also in protein biosynthesis, assembly, transport, degradation, and signal transduction (for reviews see Refs. 6 -9).HSC70, purified originally as an ATPase that uncoats clathrin-coated vesicles (10 -12), has a weak intrinsic ATPase activity (13, 14) but binds tightly to ATP and ADP (15-18). Upon binding to clathrin light chains, synthetic peptides, unfolded proteins, or other heat shock proteins such as DnaJ homologs, HSC70 ATPase activity is stimulated 2-5-fold (13, 16, 19 -28).Limited proteolysis and mutagenesis studies have shown that the protein is made of two domains, a 44-kDa aminoterminal ATPase domain that binds and hydrolyzes ATP and a 30-kDa carboxyl-terminal domain involved in the binding of th...

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

“…The binding of peptides or permanently unfolded proteins to HSC70 or DnaK promotes the dissociation of oligomers into monomers (35,46,47), suggesting that oligomerization of the protein is mediated by the peptide binding site (35). As shown in Fig.…”

“…Data analysis was performed using the computer program SVEDBERG (38) supplied by John Philo. The apparent molecular mass was determined from the sedimentation coefficient, using the relation (S 1 /S 2 ) 3 ϭ (M 1 /M 2 ) 2 and bovine serum albumin as a reference protein of known molecular mass and sedimentation coefficient (34,35,39).…”

“…Using a wide range of biophysical and biochemical techniques, we showed that HSC70 self-associates in solution, in a reversible fashion, into dimers, trimers, and probably high order oligomers, with a dissociation constant of about 5-10 M (34). Next, we analyzed the effects of the natural substrates on this equilibrium, and showed that whereas in the presence of ADP, HSC70 exists as a slow and temperature-dependent monomer-oligomer equilibrium, in the presence of ATP, peptides, or unfolded proteins, this equilibrium is shifted toward the monomer (35). Most importantly, and since the dissociation process of HSC70 oligomers into monomers appeared to be very similar to that of unfolded protein from HSC70, occurring upon ATP binding but not ATP hydrolysis, we proposed that binding of HSC70 to itself may occur via the peptide binding site and may mimic the binding of HSC70 to unfolded protein substrates (35).…”

Characterization of Interactions between the Anti-apoptotic Protein BAG-1 and Hsc70 Molecular Chaperones

“…Thus ATPase activity and substrate binding may be linked. Hsc70 self-associates into dimers, trimers, and higher order oligomers, and the oligomerization may be influenced by substrate binding, which shifts the solution state toward the monomeric species (41). Deletion of residues beyond 611 results in loss of substrate binding and may affect the stability of the overall conformation of intact Hsc70 (39).…”

Identification of proteins binding the native tubulin dimer

“…The nucleotide is bound to the N-terminal domain of HSC70 and used to drive conformational changes in the C-terminal peptide binding domain that alters its affinity for substrates [23]. In order to test a potential interaction of both nucleotide-bound forms of HSC70 with tubulin, ATP (0.5 mM) or ADP (0.5 mM) were added to the gradient before centrifugation.…”