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-domai n interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90. Limited proteolysis of recombinant HtpG, revealed three major tryptic sites , i.e. Arg7-Gly8, Ar-336-Glu337 and Lys552-Leu553, of which the latter two were located at the positions equivalent to the major cleavage sites of hum an HSP90 alpha. A similar pattern was obtained by papain treatment under no ndenaturing conditions but not under denaturing conditions. Thus, HtpG cons ists 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 expre ssed and their interactions were estimated on polyacrylamide gel electropho resis under nondenaturing conditions. As a result, two kinds of domain-doma in interactions were revealed: domain B interaction with domain A of the sa me polypeptide and domain C of one partner with domain B of the other in th e dimer. Domain B could be structurally and functionally divided into two s ubdomains, the N-terminal. two-thirds (subdomain BI) that interacted with d omain A and the C-terminal one-third (subdomain BII) that interacted with d omain C. The C-terminal two-thirds of domain A, i.e. Asp116-Arg336, were su fficient for the binding to domain B. We finally propose the domain organiz ation of an HtpG dimer.