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Bac7 is a proline-rich antimicrobial peptide, selective for Gram-negative bacteria, which acts intracellularly after membrane translocation. Progressively shortened fragments of Bac7 allowed determining the minimal sequence required for entry and antimicrobial activity as a 16-residue, N-terminal fragment, while further shortening led to a marked decrease in both functions. Furthermore, two N-terminal arginine residues were required for efficient translocation and activity. Analogues in which these residues were omitted, or where the side chain steric or physicochemical characteristics were systematically altered, were tested on different Escherichia coli strains, including a mutant with a destabilized outer membrane and one lacking the relevant SbmA membrane transport protein. H-bonding capacity, stereochemistry, and charge, in that order, played a determining role for efficient transit through both the outer and cytoplasmic membranes. Our studies allowed building a more detailed model for the mode-of-action of Bac7, and confirming its potential as an anti-infective agent, also suggesting it may be a vehicle for internalization of other antibiotic cargo.

HS1 is a protein involved in erythroid proliferation and apoptotic cell death, containing several structurally significant motifs including a C-terminal SH3 domain. HPK1 is a member of the Ste20-related kinase family, which contains four proline-rich sequences and is constitutively associated with HS1 in hematopoietic cells. Recombinant fusion protein GST-SH3(HS1) was expressed to assess the binding properties of 16 peptides derived from the HPK1 proline-rich regions. The binding affinities were determined by non-immobilized ligand interaction assay by circular dichroism. Our results revealed that the classical PxxPxK class II binding motif is not sufficient to induce the interaction with the GST-SH3(HS1) domain, an event dependent on the presence of additional basic residue(s) located at the C-terminus of the PxxPxK motif: Lys(-5) in P2 peptide and Lys(-8) in P4c peptide. Lys replacement by Arg residues decreases the ligand binding affinity. The finding that both SH3(HS1) domain and full-length HS1 protein bind to P2 peptide with similar affinity demonstrates that the whole protein sequence does not affect the interaction properties of the domain. In silico models of SH3(HS1) as a complex with P2 or P4c highlight the domain residues that interact with the recognition determinants of the peptide ligand and that cooperate in the complex stabilization.

Several aspects of the mechanism of action of Pro-rich antimicrobial peptides, together with their low toxicity ill mammalian cells, make them good candidates for the development of new antibiotic agents. We investigated the effect induced in the insect antimicrobial peptide apidaecin Ib by the replacement of a single arginine/leucine residue witha N-substituted glycine. The resulting peptoid-peptide hybrids are more resistant to proteolysis and devoid of any significant cytotoxic activity, but moving the [NArg]residue from the N- to the C-terminal end of the molecule progressively reduces the antibacterial activity. Cell uptake experiments in E. coli cells suggest that the loss of antibacterial activity of [NArg(17)]apidaecin is a consequence of its inability to translocate into bacterial cells. Conversely, apidaecin and its peptoid-peptide hybrids are able to cross the plasma membrane ill eukaryotic cells and to diffuse in the cytosol, although their translocating ability is far less effective than that of other known cell permeant peptides.