Analysis of three DnaK mutant proteins suggests that progression through the ATPase cycle requires conformational changes.
DnaK, the bacterial homolog of the eukaryotic hsp70 proteins, is an ATP-dependent chaperone whose basal ATPase is stimulated by synthetic peptides and its cohort heat shock proteins, DnaJ and GrpE. We have used three mutant DnaK proteins, E171K, D201N, and A174T (corresponding to Glu175, Asp206, and Ala179, respectively, in bovine heat stable cognate 70) to probe the ATPase cycle. All of the mutant proteins exhibit some alteration in basal ATP hydrolysis. However, they all exhibit more severe defects in the regulated activities. D201N and E171K are completely defective in all regulated activities of the protein and also in making the conformational change exhibited by the wt protein upon binding ATP. We suggest that the inability of D201N and E171K to achieve the ATP activated conformation prevents both stimulation by all effectors and the ATP-mediated release of GrpE. In contrast, the defect of A174T is much more specific. It exhibits normal binding and release of GrpE and normal stimulation of ATPase activity by DnaJ. However, it is defective in the synergistic activation of its ATPase by DnaJ and GrpE. We suggest that this mutant protein is specifically defective in a DnaJ/GrpE mediated conformational change in DnaK necessary for the synergistic action of DnaJ+GrpE.
Department of Microbiology, University of California, San Francisco 94143, USA.
A S
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Kamath-Loeb
C Z
CZ
Lu
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Suh
M A
MA
Lonetto
C A
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Gross
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D003198:Computer Simulation; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism; D006639:Histidine; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016324:Sequence Tagged Sites
D001426:Bacterial Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D010455:Peptides; D011994:Recombinant Proteins; D006639:Histidine; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli