Mutational analysis of putative helix-helix interacting GxxxG-motifs and tryptophan residues in the two-peptide bacteriocin lactococcin G.

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.