Mutagenesis reveals the complex relationships between ATPase rate and the chaperone activities of Escherichia coli heat shock protein 70 (Hsp70/DnaK).
The Escherichia coli 70-kDa heat shock protein, DnaK, is a molecular chaperone that engages in a variety of cellular activities, including the folding of proteins. During this process, DnaK binds its substrates in coordination with a catalytic ATPase cycle. Both the ATPase and protein folding activities of DnaK are stimulated by its co-chaperones, DnaJ and GrpE. However, it is not yet clear how changes in the stimulated ATPase rate of DnaK impact the folding process. In this study, we performed mutagenesis throughout the nucleotide-binding domain of DnaK to generate a collection of mutants in which the stimulated ATPase rates varied from 0.7 to 13.6 pmol/microg/min(-1). We found that this range was largely established by differences in the ability of the mutants to be stimulated by one or both of the co-chaperones. Next, we explored how changes in ATPase rate might impact refolding of denatured luciferase in vitro and found that the two activities were poorly correlated. Unexpectedly, we found several mutants that refold luciferase normally in the absence of significant ATP turnover, presumably by increasing the flexibility of DnaK. Finally, we tested whether DnaK mutants could complement growth of DeltadnaK E. coli cells under heat shock and found that the ability to refold luciferase was more predictive of in vivo activity than ATPase rate. This study provides insights into how flexibility and co-chaperone interactions affect DnaK-mediated ATP turnover and protein folding.
Chang
Lyra
L
University of Michigan, Ann Arbor, Michigan 48109, USA.
Thompson
Andrea D
AD
Ung
Peter
P
Carlson
Heather A
HA
Gestwicki
Jason E
JE
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli