title: Multistep mechanism of substrate binding determines chaperone activity of Hsp70.
abstract: The 70 kDa heat shock proteins (the Hsp70 family) assist refolding of their substrates through ATP-controlled binding. We have analyzed mutants of DnaK, an Hsp70 homolog, altered in key residues of its substrate binding domain. Substrate binding occurs by a dynamic mechanism involving: a hydrophobic pocket for a single residue that is crucial for affinity, a two-layered closing device involving independent action of an alpha-helical lid and an arch, and a superimposed allosteric mechanism of ATP-controlled opening of the substrate binding cavity that operates largely through a beta-structured subdomain. Correlative evidence from mutational analysis suggests that the ADP and ATP states of DnaK differ in the frequency of the conformational changes in the alpha-helical lid and beta-domain that cause opening of the substrate binding cavity. The affinity for substrates, as defined by this mechanism, determines the efficiency of DnaJ-mediated and ATP hydrolysis mediated locking-in of substrates and chaperone activity of DnaK.
authors.affiliation: Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Str.7, 79104 D-Freiburg, Germany.
authors.forename: M P
authors.initials: MP
authors.lastname: Mayer
authors.forename: H
authors.initials: H
authors.lastname: Schröder
authors.forename: S
authors.initials: S
authors.lastname: Rüdiger
authors.forename: K
authors.initials: K
authors.lastname: Paal
authors.forename: T
authors.initials: T
authors.lastname: Laufen
authors.forename: B
authors.initials: B
authors.lastname: Bukau
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000495:Allosteric Site / Q000187:drug effects; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004789:Enzyme Activation; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D012808:Sigma Factor; D013816:Thermodynamics; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012808:Sigma Factor; D014157:Transcription Factors; C069774:heat-shock sigma factor 32; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli