Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin.
Members of the proline-rich antibacterial peptide family, pyrrhocoricin, apidaecin and drosocin appear to kill responsive bacterial species by binding to the multihelical lid region of the bacterial DnaK protein. Pyrrhocoricin, the most potent among these peptides, is nontoxic to healthy mice, and can protect these animals from bacterial challenge. A structure-antibacterial activity study of pyrrhocoricin against Escherichia coli and Agrobacterium tumefaciens identified the N-terminal half, residues 2-10, the region responsible for inhibition of the ATPase activity, as the fragment that contains the active segment. While fluorescein-labeled versions of the native peptides entered E. coli cells, deletion of the C-terminal half of pyrrhocoricin significantly reduced the peptide's ability to enter bacterial or mammalian cells. These findings highlighted pyrrhocoricin's suitability for combating intracellular pathogens and raised the possibility that the proline-rich antibacterial peptides can deliver drug leads into mammalian cells. By observing strong relationships between the binding to a synthetic fragment of the target protein and antibacterial activities of pyrrhocoricin analogs modified at strategic positions, we further verified that DnaK was the bacterial target macromolecule. Inaddition, the antimicrobial activity spectrum of native pyrrhocoricin against 11 bacterial and fungal strains and the binding of labeled pyrrhocoricin to synthetic DnaK D-E helix fragments of the appropriate species could be correlated. Mutational analysis on a synthetic E. coli DnaK fragment identified a possible binding surface for pyrrhocoricin.
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA.
Goran
G
Kragol
Ralf
R
Hoffmann
Michael A
MA
Chattergoon
Sandor
S
Lovas
Mare
M
Cudic
Philippe
P
Bulet
Barry A
BA
Condie
K Johan
KJ
Rosengren
Luis J
LJ
Montaner
Laszlo
L
Otvos
D016960:Agrobacterium tumefaciens / Q000187:drug effects; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000187:drug effects; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D019476:Insect Proteins; D018613:Microscopy, Confocal; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D017510:Protein Folding
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D019476:Insect Proteins; C087710:pyrrhocoricin protein, Pyrrhocoris apterus; D011392:Proline; C063842:dnaK protein, E coli