D-peptides as inhibitors of the DnaK/DnaJ/GrpE chaperone system

Abstract

DnaK, a Hsp70 homolog of Escherichia coli, together with its co-chaperones DnaJ and GrpE protects denatured proteins from aggregation and promotes their refolding by an ATP-consuming mechanism. DnaJ not only stimulates the γ-phosphate cleavage of DnaK-bound ATP but also binds polypeptide substrates on its own. Unfolded polypeptides, such as denatured luciferase, thus form ternary complexes with DnaJ and DnaK. A previous study has shown that D-peptides compete with L-peptides for the same binding site in DnaJ but do not bind to DnaK (Feifel, B., Schönfeld, H.-J., and Christen, P. (1998) J. Biol. Chem. 273, 11999-12002). Here we report that D-peptides efficiently inhibit the refolding of denatured luciferase by the DnaK/DnaJ/GrpE chaperone system (EC50 = 1-2 μM). The inhibition of the chaperone action is due to the binding of D-peptide to DnaJ (Kd = 1-2 μM), which seems to preclude DnaJ from forming ternary (ATP·DnaK)m·substrate·DnaJn complexes. Apparently, simultaneous binding of DnaJ and DnaK to one and the same target polypeptide is essential for effective chaperone action.