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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Diphosphate / metabolism
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Adenosine Diphosphate / pharmacology
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Adenosine Triphosphatases / chemistry
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / metabolism
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Adenosine Triphosphate / metabolism
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Adenosine Triphosphate / pharmacology
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Allosteric Site / drug effects
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Bacterial Proteins / chemistry
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Bacterial Proteins / genetics
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Bacterial Proteins / metabolism
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Catalytic Domain
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Enzyme Activation
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Escherichia coli Proteins*
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Escherichia coli* / chemistry
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Escherichia coli* / genetics
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Escherichia coli* / metabolism
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Genetic Complementation Test
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HSP70 Heat-Shock Proteins / chemistry*
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HSP70 Heat-Shock Proteins / genetics
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HSP70 Heat-Shock Proteins / metabolism*
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Heat-Shock Proteins / metabolism
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Hydrolysis / drug effects
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Kinetics
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Models, Biological
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Models, Molecular
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Molecular Chaperones / chemistry*
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Molecular Chaperones / genetics
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Molecular Chaperones / metabolism*
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Mutation / genetics*
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Phenotype
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Protein Binding / drug effects
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Protein Structure, Secondary / drug effects
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Sigma Factor*
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Thermodynamics
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Transcription Factors / metabolism
Substances
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Bacterial Proteins
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Escherichia coli Proteins
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HSP70 Heat-Shock Proteins
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Heat-Shock Proteins
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Molecular Chaperones
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Sigma Factor
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Transcription Factors
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heat-shock sigma factor 32
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Adenosine Diphosphate
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Adenosine Triphosphate
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Adenosine Triphosphatases
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dnaK protein, E coli