IDENTIFICATION OF A REGULATORY MOTIF IN HSP7O THAT AFFECTS ATPASE ACTIVITY, SUBSTRATE-BINDING AND INTERACTION WITH HDJ-1

The Hsp70 family of molecular chaperones has an essential role in the synthesis, folding and translocation of the nascent peptide chain. Whi le the general features of these activities are well documented, less is understood about the regulation of these activities. The ATPase rat e is stimulated by non-native proteins, furthermore, interaction with ATP leads to the release of protein substrate concurrent with a confor mational change in Hsp70. One interpretation of these data is that the two domains of Hsp70 interact. In the process of mapping the carboxyl -terminal boundary of the substrate binding domain for human Hsp70, we identified a regulatory motif, EEVD, which is conserved at the extrem e carboxyl terminus among nearly all cloned cytosolic eukaryotic Hsp70 s. Deletion or mutation of EEVD affects the ATPase activity, the abili ty to interact with substrates, and interferes with the ability of the mutant Hsp70 to interact with HDJ-1 in the refolding of denatured fir efly luciferase. Examination of the biophysical properties of the muta nt Hsp70s reveals a change in the overall shape and conformation of th e protein consistent with reduced interactions between the two domains . These data suggest that the EEVD motif is involved in the intramolec ular regulation of Hsp70 function and intermolecular interactions with HDJ-1.