d-Peptide Ligands for the Co-chaperone DnaJ

The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, binds hydrophobic polypeptide segments in extended conformation. The co-chaperone DnaJ (Hsp40) has been reported to bind native and denatured proteins as well as peptides. We tested pseudo-peptides of d-amino acids as ligands for both chaperones. In comparison to the parent all-lpeptide, these mimetics had either enantiomorphic side chain positions combined with retained main chain direction (normal all-d peptide) or unchanged side chain topology together with reverse direction of the peptide backbone (retro all-d peptide). The peptides were labeled with acrylodan (a), and their binding to DnaK and DnaJ was monitored by the accompanying increase in fluorescence intensity. The parent all-l peptide a-CALLLSAARR bound to both DnaK (Kd = 0.1 μm) and DnaJ (Kd = 9.2 μm). In contrast, the normal all-dand retro all-d peptides did not bind to DnaK; they bound, however, to DnaJ withKd values of 6.8 μm and 0.9 μm, respectively. The emission spectra of the DnaJ-bound peptides suggests that DnaJ bound both d-peptides with the same main chain direction as l-peptides. Binding of thenormal all-d and all-lpeptides inhibited the DnaJ-induced stimulation of DnaK ATPase. However, binding of the retro all-d analog to DnaJ did not impair the stimulation, indicating the existence of separate binding sites for peptides and DnaK.