CHARACTERIZATION OF D10S AND K71E MUTANTS OF HUMAN CYTOSOLIC HSP70

To determine the effect of mutations at the nucleotide-binding site of recombinant Hsp70 on its interaction with protein and peptide substra tes, point mutations were made at D10 and K71, two residues at the act ive site. The D10S mutation weakened both ATP and ADP binding, while t he K71E mutation weakened only ATP binding. In binding experiments usi ng Hsp70 with no bound nucleotide, the mutated Hsp70s interacted with clathrin and peptide just like the wild-type Hsp70. However, the D10 m utation completely abolished the effects of both ATP and ADP on peptid e and clathrin binding. The K71 mutation also abolished the effect of ATP on substrate binding, but ADP, which still bound tightly, had its normal effect on substrate binding. In addition, the D10S and K71E mut ants had greatly reduced ability to uncoat clathrin-coated vesicles at pH 7.0, bind to clathrin baskets at pH 6.0, and undergo polymerizatio n induced by YDJ1 in the presence of ATP. We conclude, first, that nuc leotides must bind strongly to Hsp70 to affect substrate binding and, second, that interaction of Hsp70 with DnaJ homologues may also requir e a strongly bound ATP.