| Abstract
| - ATP binding induces a conformational change in 70-kDa heat shock proteins (Hsp70s) thatfacilitates release of bound polypeptides. Using the bovine heat shock cognate protein (Hsc70) as arepresentative of the Hsp70 family, we have characterized the effect of mutations on the coupling betweenATP binding and the nucleotide-induced conformational change. Steady-state solution small-angle X-rayscattering and kinetic fluorescence measurements on a 60-kDa fragment of Hsc70 show that point mutationsK71M, E175S, D199S, and D206S in the nucleotide binding cleft impair the ability of ATP to induce aconformational change. A secondary mutation in the peptide binding domain, E543K, “rescues” the ATP-induced transition for three of these mutations (E175S/E543K, D199S/E543K, and D206S/E543K) butnot for K71M/E543K. Analysis of kinetics of the ATPase cycle confirm that these effects do not resultfrom unexpectedly rapid ATP hydrolysis or slow ATP binding. Crystallographic structures of E175S,D199S, and D206S mutant ATPase fragment proteins show that the mutations do not perturb the tertiarystructure of the protein but do significantly alter the protein−ligand interactions, due in part to an apparentcharge compensation effect whereby mutating a (probably) negatively charged carboxyl group to a neutralserine displaces a K+ ion from the nucleotide binding cleft in two out of three cases (E175S and D199Sbut not D206S).
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