Recently, we documented that the short, proline-rich antibacterial peptides pyrrhocoricin,
drosocin, and apidaecin interact with the bacterial heat shock protein DnaK, and peptide
binding to DnaK can be correlated with antimicrobial activity. In the current report
we studied the mechanism of action of these peptides and their binding sites to Escherichia
coli DnaK. Biologically active pyrrhocoricin made of L-amino acids diminished the
ATPase activity of recombinant DnaK. The inactive D-pyrrhocoricin analogue and the
membrane-active antibacterial peptide cecropin A or magainin 2 failed to inhibit the
DnaK-mediated phosphate release from adenosine 5'-triphosphate (ATP). The effect of
pyrrhocoricin on DnaK's other significant biological function, the refolding of misfolded
proteins, was studied by assaying the alkaline phosphatase and beta-galactosidase
activity of live bacteria. Remarkably, 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 synthetic DnaK fragments
and labeled pyrrhocoricin analogues, pyrrhocoricin bound with a K(d) of 50.8 microM
to the hinge region around the C-terminal helices D and E, at the vicinity of amino
acids 583 and 615. Pyrrhocoricin binding was not observed to the homologous DnaK fragment
of Staphylococcus aureus, a pyrrhocoricin nonresponsive strain. In line with the lack
of ATPase inhibition, drosocin binding appears to be slightly shifted toward the D
helix. Our data suggest that drosocin and pyrrhocoricin binding prevents the frequent
opening and closing of the multihelical lid over the peptide-binding pocket of DnaK,
permanently closes the cavity, and inhibits chaperone-assisted protein folding. The
biochemical results were strongly supported by molecular modeling of DnaK-pyrrhocoricin
interactions. Due to the prominent sequence variations of procaryotic and eucaryotic
DnaK molecules in the multihelical lid region, our findings pave the road for the
design of strain-specific antibacterial peptides and peptidomimetics. Far-fetched
applications of the species-specific inhibition of chaperone-assisted protein folding
include the control of not only bacteria but also fungi, parasites, insects, and perhaps
rodents.