Substrate-binding characteristics of proteins in the 90 kDa heat shock protein family

In the present study we investigated the substrate-binding characteristics of three members of the 90 kDa heat shock protein (HSP90) family, namely th e cr isoform of human HSP90 (HSP90 alpha), human GRP94 (94 kDa glucose-regu lated protein, a form of HSP90 from endoplasmic reticulum), and HtpG (the E scherichia coli homologue of HSP90) and the domain responsible for these ch aracteristics. The recombinant forms of HSP90a, GRP94 and HtpG existed as d imers and became oligomerized at higher temperatures. Among the three famil y members, HtpG required the highest temperature (65 degreesC) for its tran sition to oligomeric forms. The precipitation of the substrate protein, glu tathione S-transferase, which occurred at 55 degreesC, was efficiently prev ented by the simultaneous presence of a sufficient amount of HSP90 alpha or GRP94, but not by HtpG, which was still present as a dimer at that tempera ture. However, precipitation was stopped completely at 65-70 degreesC, at w hich temperature HtpG was oligomerized. Thus the transition of HSP90-family proteins to a state with self-oligomerization ability is essential for pre venting the precipitation of substrate proteins. We then investigated the d omain responsible for the substrate binding of HtpG on the basis of the thr ee domain structures. The self-oligomerizing and substrate-binding activiti es towards glutathione S-transferase and citrate synthase were both located in a single domain, the N-terminal domain (residues 1-336) of HtpG. We the refore propose that the primary peptide-binding site is located in the N-te rminal domain of HSP90-family proteins.