A disulfide-bonded DnaK dimer is maintained in an ATP-bound state

Area of Science:

• Molecular Biology
• Protein Structure and Function
• Biochemistry

Background:

• DnaK, a key Hsp70 chaperone in E. coli, is a model for Hsp70 research.
• Crystal structure revealed DnaK forms a dimer with ATP.
• Previous studies suggested this dimer is vital for Hsp40 co-chaperone interaction.

Purpose of the Study:

• To investigate the biochemical properties of the DnaK dimer.
• To create a stable DnaK dimer to analyze its function.
• To understand the role of DnaK dimer dynamics in the chaperone cycle and Hsp40 interaction.

Main Methods:

• Site-directed mutagenesis (A303C, H541C) to create a disulfide-bond-linked DnaK dimer.
• Oxidation to form the specific DnaK dimer in the presence of ATP.
• Biochemical assays to measure ATPase activity, peptide binding kinetics, and Hsp40 interaction.

Main Results:

• The disulfide-bond-linked DnaK dimer exhibited reduced ATPase activity and peptide-binding affinity.
• The dimer showed accelerated peptide binding even without ATP, indicating a locked ATP-bound state.
• Interaction with Hsp40 co-chaperone was significantly compromised in the disulfide-linked DnaK dimer.

Conclusions:

• The DnaK dimer, while transiently formed, can become locked in an ATP-bound state, inhibiting the chaperone cycle.
• Dissociation of the DnaK dimer is essential for progression through the chaperone cycle.
• DnaK dimer dissociation is critical for efficient Hsp40 co-chaperone interaction, enabling stable Hsp70-Hsp40 complex formation.