The Antibacterial Peptide Pyrrhocoricin Inhibits the ATPase Actions of DnaK and Prevents Chaperone-Assisted Protein Folding

Abstract

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.

The insect antimicrobial peptide, -pyrrhocoricin, binds to and stimulates the ATPase activity of both wild-type and lidless DnaK

“…The main findings from previous studies [4,20] of PYRDnaK interactions are summarized as follows: (i) L L -PYR inhibits the ATPase activity of DnaK; (ii) biotinylated-L L -PYR specifically binds to DnaK (590-615), which is a fragment containing the aD-aE helices; (iii) biotinylated-L L -PYR nonspecifically binds to both DnaK (397-439), which is a fragment of the substrate-binding domain, and to DnaK (596-637), which constitutes the aE helix and the 30-residue flexible Cterminus; and (iv) the N-terminal residues 1-9 of L L -PYR tightly bind to an 'allosteric ATPase site' on DnaK [20], which explains the ability of L L -PYR to inhibit DnaK-mediated ATP hydrolysis. Aspects of these earlier findings and their relation to our findings are discussed below.…”

“…The image was constructed from PDB file 1DKX. L L -PYR inhibits DnaK's ATPase activity [20]. Because L L -PYR binds to ATP-bound wild-type and lidless DnaK like other short peptides, we tested whether L L -PYR stimulates the ATPase activity of both forms of DnaK using a single turnover assay ([DnaK or DnaK(2-517)] ) [ATP]).…”

“…It has been hypothesized that proline-rich antibacterial peptides kill bacteria by preventing the frequent movements of the multi-helical lid over the substrate-binding site [20]. We sought to test this hypothesis using the following postulate as a basis for the experimental design: If DnaK possesses two peptide binding sites, then it is possible that DnaK forms a two-peptide complex in which a short fluorescently tagged peptide molecule, such as aNR or ap5, is bound in the conventional substrate-binding site and an L L -PYR molecule is bound to the lid.…”

“…The question is whether DnaK contains such an allosteric ATPase site. On the basis of dot blot binding experiments and computer modeling, Kragol et al [20] concluded that PYR binds to the aD + aE helices, and because binding down regulated DnaK's ATPase activity, they hypothesized that the aD + aE helices are the allosteric ATPase site. Their hypothesis was reinforced by a recent study showing that the ATPase activity of DnaK is allosterically modulated by the C-terminal lid domain, particularly by the aD + aE helices [24].…”

Ultrastructural observation on sterilization of melittin

“…Benachir considered that the affinity of melittin for phosphatidylcholine vesicles was responsible for the increasing melittin-induced leakage from cholesterolcontaining membranes, and an allor-none hypothesis was proposed [3]. The Shai-MatsuzakiHuang (SMH) model proposed that the interaction of the peptide with the membrane was followed by the displacement of lipids [4][5][6][7][8]. Five hypotheses concerning the possible mechanisms for antimicrobial peptide action [3] were summarized in Nature.…”