title: 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.
authors.affiliation: Department of Molecular Biosciences, University of Oslo, Pb 1041 Blindern, 0316 Oslo, Norway. camilla.oppegard@imbv.uio.no
authors.forename: Camilla
authors.initials: C
authors.lastname: Oppegård
authors.forename: Juliane
authors.initials: J
authors.lastname: Schmidt
authors.forename: Per Eugen
authors.initials: PE
authors.lastname: Kristiansen
authors.forename: Jon
authors.initials: J
authors.lastname: Nissen-Meyer
mesh_terms: D020816:Amino Acid Motifs; D001430:Bacteriocins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007778:Lactobacillus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
chemical_list: D001430:Bacteriocins; D010455:Peptides; C110915:lactococcin G; D014364:Tryptophan