title: Domain-domain interactions of HtpG, an Escherichia coli homologue of eukaryotic HSP90 molecular chaperone.
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.
authors.affiliation: Department of Oral Biochemistry, Nagasaki University School of Dentistry, Nagasaki, Japan. tnemoto@net.nagasaki-u.ac.jp
authors.forename: T K
authors.initials: TK
authors.lastname: Nemoto
authors.forename: T
authors.initials: T
authors.lastname: Ono
authors.forename: T
authors.initials: T
authors.lastname: Kobayakawa
authors.forename: E
authors.initials: E
authors.lastname: Tanaka
authors.forename: T T
authors.initials: TT
authors.lastname: Baba
authors.forename: K
authors.initials: K
authors.lastname: Tanaka
authors.forename: T
authors.initials: T
authors.lastname: Takagi
authors.forename: T
authors.initials: T
authors.lastname: Gotoh
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D019281:Dimerization; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D006639:Histidine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018841:HSP90 Heat-Shock Proteins; C489352:HtpG protein, E coli; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; C066720:HtpG protein, bacteria; C033223:polyhistidine; D006639:Histidine; D014357:Trypsin; D010206:Papain