Interdomain communication in the molecular chaperone DnaK

Area of Science:

• Molecular Biology
• Biochemistry
• Protein Dynamics

Background:

• DnaK, an Escherichia coli Hsp70 homologue, has a single tryptophan in its ATPase domain.
• Intrinsic fluorescence of this tryptophan is sensitive to ligand binding and conformational changes.
• Understanding DnaK's conformational states is crucial for its chaperone function.

Purpose of the Study:

• To investigate the conformational changes in DnaK upon binding of ATP, ADP, GrpE, and peptides.
• To correlate fluorescence changes with specific domain conformations (ATPase and substrate-binding).
• To elucidate the kinetics of ligand binding and its effect on DnaK's overall structure.

Main Methods:

• Utilized intrinsic tryptophan fluorescence spectroscopy of DnaK.
• Monitored fluorescence changes upon addition of ATP, ADP plus GrpE, and peptides.
• Analyzed differential kinetics of peptide binding to various DnaK-ligand complexes.

Main Results:

• ATP or ADP/GrpE binding induced a similar decrease in fluorescence, suggesting an open ATPase domain conformation.
• Peptide binding increased fluorescence for both ATP.DnaK and ADP.DnaK.GrpE, indicating a closed ATPase domain.
• Differential peptide binding kinetics were observed between ADP.DnaK and ATP.DnaK/ADP.DnaK.GrpE complexes.

Conclusions:

• Ligand binding to the ATPase domain (ATP or ADP/GrpE) and substrate-binding domain (peptides) influences DnaK's conformation.
• Conformational shifts towards open and closed states occur in a concerted manner.
• These findings provide insights into the allosteric regulation of DnaK function.