title: Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication.
abstract: Interactions of the DnaK (Hsp70) chaperone from Escherichia coli with substrates are controlled by ATP. Nucleotide-induced changes in DnaK conformation were investigated by monitoring changes in tryptic digestion pattern and tryptophan fluorescence. Using nucleotide-free DnaK preparations, not only the known ATP-induced major changes in kinetics and pattern of proteolysis but also minor ADP-induced changes were detected. Similar ATP-induced conformational changes occurred in the DnaK-T199A mutant protein defective in ATPase activity, demonstrating that they result from binding, not hydrolysis, of ATP. N-terminal sequencing and immunological mapping of tryptic fragments of DnaK identified cleavage sites that, upon ATP addition, appeared within the proposed C-terminal substrate binding region and disappeared in the N-terminal ATPase domain. They hence reflect structural alterations in DnaK correlated to substrate release and indicate ATP-dependent domain interactions. Domain interactions are a prerequisite for efficient tryptic degradation as fragments of DnaK comprising the ATPase and C-terminal domains were highly protease-resistant. Fluorescence analysis of the N-terminally located single tryptophan residue of DnaK revealed that the known ATP-induced alteration of the emission spectrum, proposed to result directly from conformational changes in the ATPase domain, requires the presence of the C-terminal domain and therefore mainly results from altered domain interaction. Analyses of the C-terminally truncated DnaK163 mutant protein revealed that nucleotide-dependent interdomain communication requires a 15-kDa segment assumed to constitute the substrate binding site.
authors.affiliation: Zentrum für Molekulare Biologie, Universität Heidelberg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Buchberger
authors.forename: H
authors.initials: H
authors.lastname: Theyssen
authors.forename: H
authors.initials: H
authors.lastname: Schröder
authors.forename: J S
authors.initials: JS
authors.lastname: McCarty
authors.forename: G
authors.initials: G
authors.lastname: Virgallita
authors.forename: P
authors.initials: P
authors.lastname: Milkereit
authors.forename: J
authors.initials: J
authors.lastname: Reinstein
authors.forename: B
authors.initials: B
authors.lastname: Bukau
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D029968:Escherichia coli Proteins; D005453:Fluorescence; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli