Physical interactions between members of the DnaK chaperone machinery: characterization of the DnaK.GrpE complex.
Many of the functions of the Escherichia coli Hsp70, Dnak, require two cofactors, DnaJ and GrpE. GrpE acts as a nucleotide exchange factor in the DnaK reaction cycle but the details of its mechanism remain unclear. GrpE has high affinity for monomeric native DnaK, with a Kd estimated at < or = 50 nM. GrpE is a very asymmetric molecule and exists as either a dimer or trimer in its native state. The stoichiometry of GrpE to DnaK in the isolated complex was 3:1, suggesting a trimer. Formation of the complex is quite fast (kan > 1 s-1), whereas the off-rate is very slow on the HPLC timescale (kant < or = 10(-4) s-1). GrpE has no affinity for ATP or ADP, nor the oligomeric and molten globule states of DnaK. The complex is much more thermally stable than either GrpE or DnaK alone, and prevents the formation of the molten globule-like state of DnaK at physiologically relevant temperatures. Formation of the complex does not cause any change in secondary structure, as determined by the lack of change in the circular dichroism spectrum. However, binding of GrpE induces a similar tertiary structural change in DnaK to that induced by binding of ATP, based on the blue shift in lambda max from the fluorescence of the single tryptophan in DnaK. The nucleotide exchange properties of GrpE can be explained by the conformational change which may represent the opening of the nucleotide cleft on DnaK, subsequently inducing a low affinity state for ADP.
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Department of Chemistry and Biochemistry, University of California, Santa Cruz 95064, USA.
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004355:Drug Stability; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism*; D006361:Heating; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli