IN-VITRO DISSOCIATION OF BIP-PEPTIDE COMPLEXES REQUIRES A CONFORMATIONAL CHANGE IN BIP AFTER ATP BINDING BUT DOES NOT REQUIRE ATP HYDROLYSIS

In the present study, we produced single point mutations in the ATP bi nding site of hamster Rip, isolated recombinant proteins, and characte rized them in terms of their affinity for ATP and ADP, their ability t o undergo a conformational change upon nucleotide binding, and their r ate of ATP hydrolysis, These analyses allowed us to classify the mutan ts into three groups: ATP hydrolysis (T229G), ATP binding (G226D, G227 D), and ATP-induced conformation (T37G) mutants, and to test the role of these activities in the in vitro ATP-mediated release of proteins f rom Rip. All three classes of mutants were still able to bind peptide demonstrating that nucleotide is not involved in this function, Additi on of ATP to either wild-type BiP or the T229G mutant caused the in vi tro release of bound peptide, confirming that ATP hydrolysis is not re quired for protein release. ATP did not dissociate G226D, G227D, or T3 7G mutant BiP-peptide complexes, suggesting that ATP binding to Rip is not sufficient for the release of bound peptides, but that an ATP-ind uced conformational change in BiP is necessary, The identification of BiP mutants that are defective in each of these steps of ATP hydrolysi s will allow the in vivo dissection of the role of nucleotide in BiP's activity.