Identification of crucial residues for the antibacterial activity of the proline‐rich peptide, pyrrhocoricin

Abstract

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.

Flow cytometric analysis of the contributing factors for antimicrobial activity enhancement of cell-penetrating type peptides: Case study on engineered apidaecins

“…These results suggested that the N-terminal region of apidaecin contributed to both the cell-penetration and inhibition of target molecule(s). In fact, the N-terminus of apidaecin has been proposed as an inhibitor domain interacting with intracellular targets by analogy with pyrrhocoricin, in which the C-terminally truncated peptide interacted with its target, DnaK [8,19]. The results in the present study supported this hypothesis.…”

“…The activities of the peptides were decreased by FAM-labeling, as observed for the case of pyrrhocoricin [10,19]. However, the two engineered apidaecins, 1G-17 and 1C-20, with FAM exhibited higher activity compared to the labeled wild-type peptide, indicating that the FAM-labeling did not impair the activity enhancement of the engineered peptides.…”

“…Mutagenesis studies previously demonstrated that the C-terminal half of apidaecin was essential for antimicrobial activity [11,18], thus modification of the C-terminal likely inactivates the peptides. Additionally, N-terminally FAM-labeled pyrrhocoricin, a proline-rich AMP, in which a Lys residue was 8 inserted as a spacer between the N-terminus of peptide and the labeled substance, retained antimicrobial activity [19]. Therefore, we used the N-terminal α-amino group of the extra Lys as a labeling site (Table 1).…”

Primary Structure and in Vitro Antibacterial Properties of the Drosophila melanogaster Attacin C Pro-domain

“…For example, pyrrhocoricin was shown to inhibit protein folding in bacteria by inactivating the multihelical lid complex of the 70-kDa heat shock protein DnaK needed for substrate binding and ATPase activity (29). All these effects, however, require an initial interaction with the bacterial membrane followed by a membrane penetration step (34), often inefficient for the short, proline-rich antimicrobial peptide family. Therefore, we have investigated possible synergistic interactions between MPAC and cecropin A, another antimicrobial peptide known to increase plasma membrane permeability, which could facilitate MPAC entrance into bacterial cells.…”

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

“…According to recent reports, L L -PYR binds to wild-type DnaK at two sites, the conventional substrate-binding site and the aD and aE helices of the multi-helical lid [4,18]. In the experiments described below the binding of L L -PYR to the conventional substrate-binding site of DnaK was characterized.…”