Mutations in the DnaK chaperone affecting interaction with the DnaJ cochaperone

Area of Science:

• Molecular Biology
• Protein Folding
• Chaperone Proteins

Background:

• Heat shock protein 70 (Hsp70) chaperones are essential for protein homeostasis, utilizing ATP-dependent cycles to bind and release substrates.
• ATP hydrolysis is a critical, rate-limiting step in the Hsp70 ATPase cycle, leading to substrate sequestration.
• DnaJ cochaperones are known to significantly accelerate this ATP hydrolysis step.

Purpose of the Study:

• To investigate the specific requirements of the Escherichia coli Hsp70 homolog, DnaK, for stimulation by the DnaJ cochaperone.
• To identify the structural domains of DnaK involved in the functional interaction with DnaJ.
• To elucidate the mechanism by which DnaJ couples substrate binding to ATP hydrolysis in DnaK.

Main Methods:

• Mutagenesis of the ATPase domain of DnaK to probe functional interactions with DnaJ.
• Analysis of the effects of mutations on DnaK's ATPase activity and substrate binding in the presence of DnaJ.
• Comparative analysis of conserved structural elements between DnaK and other Hsp70s, and DnaJ and its homologs.

Main Results:

• The stimulation of DnaK's ATPase activity by DnaJ requires the intact, linked ATPase and substrate-binding domains of DnaK.
• Mutations within an exposed channel in the DnaK ATPase domain impair the functional interaction with DnaJ.
• This channel is proposed as the binding site for DnaJ, facilitating the coupling of substrate binding to ATP hydrolysis.

Conclusions:

• DnaJ cochaperones stimulate Hsp70 ATPase activity by interacting with a specific channel in the Hsp70 ATPase domain.
• This interaction is crucial for coupling substrate binding to the rate-limiting ATP hydrolysis step.
• The evolutionary conservation of this channel and the DnaJ J-domain suggests a conserved mechanism for Hsp70-cochaperone function across species.