D-peptide ligands for the co-chaperone DnaJ

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Folding

Background:

• The molecular chaperone DnaK, an Hsp70 homolog in Escherichia coli, recognizes and binds hydrophobic polypeptide segments.
• The co-chaperone DnaJ (Hsp40) interacts with both native and denatured proteins, as well as peptides.

Purpose of the Study:

• To investigate the binding specificities of DnaK and DnaJ for peptides composed of D-amino acids.
• To elucidate the role of peptide backbone direction and side chain topology in chaperone-peptide interactions.

Main Methods:

• Synthesis and fluorescent labeling (with acrylodan) of all-L, normal all-D, and retro all-D pseudo-peptides.
• Fluorescence spectroscopy to monitor peptide binding to DnaK and DnaJ.
• Assay of DnaJ-stimulated DnaK ATPase activity.

Main Results:

• The all-L peptide bound to both DnaK (Kd = 0.1 μM) and DnaJ (Kd = 9.2 μM).
• Neither the normal all-D nor the retro all-D peptides bound to DnaK.
• Both D-peptide analogs bound to DnaJ (Kd = 6.8 μM and 0.9 μM, respectively), with spectral data suggesting conserved main chain direction.
• Binding of normal all-D and all-L peptides inhibited DnaJ-stimulated DnaK ATPase, but retro all-D peptide binding did not.

Conclusions:

• DnaK exhibits strict specificity for L-peptides, while DnaJ can bind both L- and D-peptides.
• The binding of D-peptides to DnaJ suggests distinct binding sites for peptides and DnaK.
• These findings highlight differential substrate recognition mechanisms between DnaK and DnaJ.