PMID:11606187
| Citation |
Nemoto, TK, Ono, T, Kobayakawa, T, Tanaka, E, Baba, TT, Tanaka, K, Takagi, T and Gotoh, T (2001) Domain-domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90 molecular chaperone. Eur. J. Biochem. 268:5258-69 |
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| Abstract |
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 HSP90alpha. 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. |
| Links | |
| Keywords |
Amino Acid Sequence; Bacterial Proteins; Dimerization; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Escherichia coli/chemistry; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; HSP90 Heat-Shock Proteins/chemistry; HSP90 Heat-Shock Proteins/metabolism; Histidine; Molecular Sequence Data; Molecular Weight; Papain/metabolism; Peptide Fragments/chemistry; Peptide Fragments/metabolism; Peptides/metabolism; Protein Binding; Protein Interaction Mapping; Protein Structure, Tertiary; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Substrate Specificity; Trypsin/metabolism |
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