CONFORMATIONS OF THE NUCLEOTIDE AND POLYPEPTIDE BINDING DOMAINS OF A CYTOSOLIC HSP70 MOLECULAR CHAPERONE ARE COUPLED

70-kDa heat shock protein (Hsp70) molecular chaperones are ATPases tha t participate in protein folding by regulating protein-protein interac tions. ATP binds to the highly conserved amino-terminal domain, wherea s polypeptides bind to the less conserved carboxyl-terminal domain. Th ese domains are functionally coupled. Polypeptides were previously sho wn to dissociate from Hsp70s upon ATP binding and to stimulate ATPase activity. We probed the structure of the yeast cytosolic Hsp70 Ssa1p u sing limited proteolysis to determine whether the conformations of its nucleotide and polypeptide binding domains are also coupled. Ssa1p ad opted three distinct conformations, nucleotide-free, ADP-dependent, an d ATP-dependent. Complete conformational changes required K+ and Mg2+. Using amino-terminal sequencing, ATP-agarose chromatography, and a ca rboxyl-terminal-specific antibody, we mapped the locations of the majo r proteolytic fragments. Nucleotides altered the conformations of both the nucleotide and polypeptide binding domains. Similarly, a polypept ide altered the conformations of both domains. These results indicate that the conformations of the nucleotide and polypeptide binding domai ns are coupled.