Mutagenesis Reveals the Complex Relationships between ATPase Rate and the Chaperone Activities of Escherichia coli Heat Shock Protein 70 (Hsp70/DnaK)

Lyra Chang,Andrea D Thompson,Peter Ung,Heather A Carlson,Jason E Gestwicki

doi:10.1074/jbc.m110.124149

Lyra Chang, Andrea D Thompson + Show 3 more

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https://doi.org/10.1074/jbc.m110.124149

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Abstract

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.

Highlights

One of the main roles of DnaK is to enable the folding of nascent or otherwise unfolded proteins [6]
The nucleotide-binding domain (NBD) of DnaK is further divided into four subdomains as follows: IA/IIA, which form the base, and IB/IIB, which form the upper walls of the nucleotide binding cleft (Fig. 1A)
Design of DnaK Mutants—In this study we aimed to assess whether changes in the ATPase rate of DnaK lead to predictable changes in chaperone function

Summary

Introduction

One of the main roles of DnaK is to enable the folding of nascent or otherwise unfolded proteins [6]. GrpE, on the other hand, induces nucleotide exchange and leads to substrate release [21] These co-chaperone activities appear to be required for the cellular functions of DnaK because deletion of either DnaJ or GrpE causes defects in growth at elevated temperatures, similar to what is seen in ⌬dnaK strains [5, 22, 23]. It was found that DnaK requires DnaJ and GrpE to refold denatured luciferase [6] This platform has been used to explore the roles of ATP hydrolysis in controlling substrate folding. Truncated forms of DnaJ, which are able to stimulate ATP hydrolysis normally but cannot interact with substrates, are unable to stimulate luciferase refolding [18] Together, these results suggest that ATPase activity is necessary but not sufficient to achieve luciferase folding. Similar to what was observed in the in vitro luciferase refolding experiments, the ATPase activity of DnaK appears to be required during heat shock, because active site mutations that abolish nucleotide turnover are unable to rescue heat shock (24 –26)

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