The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding

Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin, drosocin, and apidaecin interact with the bacterial heat sh ock protein DnaK, and peptide binding to DnaK can be correlated with antimi crobial activity. In the current report we studied the mechanism of action of these peptides and their binding sites to Escherichia coli DnaK. Biologi cally active pyrrhocoricin made of L-amino acids diminished the ATPase acti vity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the mem brane-active antibacterial peptide cecropin A or magainin 2 failed to inhib it the DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP) . The effect of pyrrhocoricin on DnaK's other significant biological functi on, the refolding of misfolded proteins, was studied by assaying the alkali ne phosphatase and beta -galactosidase activity of live bacteria. Remarkabl y, both enzyme activities were reduced upon incubation with L-pyrrhocoricin or drosocin. D-Pyrrhocoricin, magainin 2, or buforin II, an antimicrobial peptide involved in binding to bacterial nucleic acids, had only negligible effect. According to fluorescence polarization and dot blot analysis of sy nthetic DnaK fragments and labeled pyrrhocoricin analogues, pyrrhocoricin b ound with a K-d of 50.8 muM to the hinge region around the C-terminal helic es D and E, at the vicinity of amino acids 583 and 615. Pyrrhocoricin bindi ng was not observed to the homologous DnaK fragment of Staphylococcus aureu s, a pyrrhocoricin nonresponsive strain. In line with the lack of ATPase in hibition, drosocin binding appears to be slightly shifted toward the D heli x. Our data suggest that drosocin and pyrrhocoricin binding prevents the fr equent opening and closing of the multihelical lid over the peptide-binding pocket of DnaK, permanently closes the cavity, and inhibits chaperone-assi sted protein folding. The biochemical results were strongly supported by mo lecular modeling of DnaK-pyrrhocoricin interactions. Due to the prominent s equence variations of procaryotic and eucaryotic DnaK molecules in the mult ihelical lid region, our findings pave the road for the design of strain-sp ecific antibacterial peptides and peptidomimetics. Far-fetched applications of the species-specific inhibition of chaperone-assisted protein folding i nclude the control of not only bacteria but also fungi, parasites, insects, and perhaps rodents.