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

G. Kragol, Sándor Lovas, G. Varadi, B. A. Condie, R. Hoffmann, L. Otvos

Research output: Contribution to journalArticle

300 Citations (Scopus)

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 β-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 Kd of 50.8 μM 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.

Original languageEnglish
Pages (from-to)3016-3026
Number of pages11
JournalBiochemistry
Volume40
Issue number10
DOIs
StatePublished - Mar 13 2001

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Protein folding
Protein Folding
Adenosine Triphosphatases
Peptides
Magainins
Pyrrhocoris apterus pyrrhocoricin protein
Bacteria
Galactosidases
Protein Refolding
Peptidomimetics
Amino Acids
Fluorescence Polarization
Molecular modeling
Bacterial Proteins
Enzyme activity
Hinges
Heat-Shock Proteins
Fungi
Proline
Adenosine

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. / Kragol, G.; Lovas, Sándor; Varadi, G.; Condie, B. A.; Hoffmann, R.; Otvos, L.

In: Biochemistry, Vol. 40, No. 10, 13.03.2001, p. 3016-3026.

Research output: Contribution to journalArticle

Kragol, G. ; Lovas, Sándor ; Varadi, G. ; Condie, B. A. ; Hoffmann, R. ; Otvos, L. / The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. In: Biochemistry. 2001 ; Vol. 40, No. 10. pp. 3016-3026.
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AU - Otvos, L.

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