DESTABILIZATION OF PEPTIDE BINDING AND INTERDOMAIN COMMUNICATION BY AN E543K MUTATION IN THE BOVINE 70-KDA HEAT-SHOCK COGNATE PROTEIN, A MOLECULAR CHAPERONE

We have compared 70-kDa heat shock cognate protein (Hsc70) isolated fr om bovine brain with recombinant wild type protein and mutant E543K pr otein (previously studied as wild type in our laboratory), Wild type b ovine and recombinant protein differ by posttranslational modification of lysine 561 but interact similarly with a short peptide (fluorescei n-labeled FYQLALT) and with denatured staphylococcal nuclease-(Delta 1 35-149). Mutation E543K results in 4.5-fold faster release of peptide and lower stability of complexes with staphylococcal nuclease-(Delta 1 35-149). ATP hydrolysis rates of the wild type proteins are enhanced 6 -10-fold by the addition of peptide. The E543K mutant has a peptide-st imulated hydrolytic rate similar to that of wild type protein but a hi gher unstimulated rate, yielding a mere 2-fold enhancement. All three versions of Hsc70 possess similar ATP-dependent conformational shifts, and all show potassium ion dependence. These data support the followi ng model: (i) in the presence of K+, Mg2+, and ATP, the peptide bindin g domain inhibits the ATPase; (ii) binding of peptide relieves this in hibition; and (iii) the E543K mutation significantly attenuates the in hibition by the peptide binding domain and destabilizes Hsc70-peptide complexes.