Mutational Analysis of Putative Helix−Helix Interacting GxxxG-Motifs and Tryptophan Residues in the Two-Peptide Bacteriocin Lactococcin G

Abstract

The membrane-permeabilizing two-peptide bacteriocin lactococcin G consists of two different peptides, LcnG-alpha and LcnG-beta. The bacteriocin contains several tryptophan and tyrosine residues and three putative helix-helix interacting GxxxG-motifs, G 7xxxG 11 and G 18xxxG 22 in LcnG-alpha and G 18xxxG 22 in LcnG-beta. The tryptophan and tyrosine residues and residues in the GxxxG-motifs were altered by site-directed mutagenesis to analyze the structure and membrane-orientation of lactococcin G. Substituting the glycine residues at position 7 or 11 in the G 7xxxG 11-motif in LcnG-alpha with large hydrophobic or hydrophilic residues was highly detrimental, whereas small residues were tolerated. Qualitatively similar results were obtained for the G 18xxxG 22-motif in LcnG-beta. In contrast, replacement of the glycine residues in the middle of these two motifs with large hydrophilic residues was tolerated. All mutations in the G 18xxxG 22-motif in LcnG-alpha were relatively well-tolerated, indicating that this motif is not involved in helix-helix interactions. The four aromatic residues in the N-terminal part of LcnG-beta could individually be replaced by other aromatic residues, a hydrophilic positive residue, and a hydrophobic residue without a marked reduced activity, indicating that this region is structurally flexible and not embedded in a strictly hydrophobic or hydrophilic environment. The results are in accordance with a structural model where the G 7xxxG 11-motif in LcnG-alpha and the G 18xxxG 22-motif in LcnG-beta interact and allow the two peptides to form a parallel transmembrane helix-helix structure, with the tryptophan-rich N-terminal part of LcnG-beta positioned in the outer membrane interface and the cationic C-terminal end of LcnG-alpha inside the cell.

The Lactococcin G Immunity Protein Recognizes Specific Regions in Both Peptides Constituting the Two-Peptide Bacteriocin Lactococcin G

“…It has been speculated previously that the two-peptide bacteriocins recognize target cells through a receptor in the target cell membrane (26). Recently, it was shown that a component of the mannose phosphotransferase system (mannose-PTS) is the target cell receptor for several class II bacteriocins such as lactococcin A and the pediocin-like bacteriocins (9).…”

“…Lactococcin G is perhaps the best-characterized two-peptide bacteriocin (12,18,19,21,24,26,27). It consists of the 39-residue ␣ peptide (termed Lcn-␣) and the 35-residue ␤ peptide (termed Lcn-␤) (Fig.…”

“…Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy have revealed that the two lactococcin G peptides adopt mainly ␣-helical structures when they are individually exposed to membrane-like entities (12,27). Based on the NMR structures and findings from site-directed mutagenesis studies, a structural model of lactococcin G has recently been proposed (23,26,27). In this model, the two complementary peptides form parallel helices that span the target cell membrane.…”

Synthesis of Trypsin-Resistant Variants of the Listeria -Active Bacteriocin Salivaricin P

“…For both class I and class IIa bacteriocins, these residues have frequently been found to play an important role in mediating the initial interaction with the anionic target cells via electrostatic interactions (3,4,17). Indeed, recent studies with the most extensively studied class IIb bacteriocin, lactococcin G, also revealed an important role for such residues in positioning the cationic C termini of the bacteriocin components inside the target cell (23,25,27). Here, we sought to maintain the potent antilisterial activity of the salivaricin P components while establishing a trypsin-resistant phenotype.…”

“…Theoretically, engineering of a bacteriocin to reduce its sensitivity to proteases is also a viable option. Engineering of bacteriocins has been used to generate variants which have helped scientists to better understand the structure-function relationships of these peptides (6,7,10,11,14,24,25,28,31,34), and there have been recent successes in which the antimicrobial activity of bacteriocins has been enhanced through a bioengineering-based approach (1,8,9,15,17,19,35). Despite this, bioengineered peptides with enhanced protease resistance have been generated only in the case of the class I bacteriocin gallidermin.…”