Domain–domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90 molecular chaperone

In the present study, we investigated the domain structure and domain–domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90. Limited proteolysis of recombinant HtpG, revealed three major tryptic sites, i.e. Arg7‐Gly8, Arg336‐Glu337 and Lys552‐Leu553, of which the latter two were located at the positions equivalent to the major cleavage sites of human HSP90α. A similar pattern was obtained by papain treatment under nondenaturing conditions but not under denaturing conditions. Thus, HtpG consists of three domains, i.e. Domain A, Met1–Arg336; domain B, Glu337–Lys552; and domain C, Leu553‐Ser624, as does HSP90. The domains of HtpG were expressed and their interactions were estimated on polyacrylamide gel electrophoresis under nondenaturing conditions. As a result, two kinds of domain–domain interactions were revealed: domain B interaction with domain A of the same polypeptide and domain C of one partner with domain B of the other in the dimer. Domain B could be structurally and functionally divided into two subdomains, the N‐terminal two‐thirds (subdomain BI) that interacted with domain A and the C‐terminal one‐third (subdomain BII) that interacted with domain C. The C‐terminal two‐thirds of domain A, i.e. Asp116–Arg336, were sufficient for the binding to domain B. We finally propose the domain organization of an HtpG dimer.