Structure analysis of the two-peptide bacteriocin lactococcin G by introducing d-amino acid residues

Abstract

The importance of 3D structuring in the N-and C-terminal ends of the two peptides (39-mer LcnG-a and 35-mer LcnG-b) that constitute the two-peptide bacteriocin lactococcin G was analysed by replacing residues in the end regions with the corresponding D-isomeric residues. When assayed for antibacterial activity in combination with the complementary wild-type peptide, LcnG-a with four D-residues in its C-terminal region and LcnG-b with four D-residues in either its N-or its C-terminal region were relatively active (two-to 20-fold reduction in activity). 3D structuring of the C-terminal region in LcnG-a and the C-and N-terminal regions in LcnG-b is thus not particularly critical for retaining antibacterial activity, indicating that the 3D structure of these regions is not vital for interpeptide interactions or for interactions between the peptides and cellular components. The 3D structure of the N-terminal region in LcnG-a may be more important, as LcnG-a with four N-terminal D-residues was the least active of these four peptides (10-to 100-fold reduction in activity). The results are consistent with a proposed structural model of lactococcin G in which LcnG-a and -b form a transmembrane parallel helix-helix structure involving approximately 20 residues in each peptide, starting near the N terminus of LcnG-a and at about residue 13 in LcnG-b. Upon expressing the lactococcin G immunity protein, sensitive target cells became resistant to all of these D-residue-containing peptides. The end regions of the two lactococcin G peptides are consequently not involved in essential structure-dependent interactions with the immunity protein. The relatively high activity of most of the D-residuecontaining peptides suggests that bacteriocins with increased resistance to exopeptidases may be generated by replacing their N-and C-terminal residues with D-residues.

Defining the Structure and Receptor Binding Domain of the Leaderless Bacteriocin LsbB

“…In the case of the two-peptide bacteriocin lactococcin G, one of the peptides (LcnG-␣), like LsbB, contains a series of cationic residues (residues 35-39: RKKKH) at the C-terminal part. This feature is believed to function as a means to force the peptide through the target cell membrane by the membrane potential (39). Whether this is the case also for LsbB with regard to this basic feature awaits further investigation.…”

Sensitivity to the two‐peptide bacteriocin lactococcin G is dependent on UppP, an enzyme involved in cell‐wall synthesis

“…The alpha and beta peptides of lactococcin G and enterocin 1071 were purified from a two‐plasmid expression system in L. sakei Lb790 as previously described (Oppegård et al ., 2007; 2010).…”

“…Lactococcin G consists of the 39‐residue alpha‐peptide and the 35‐residue beta‐peptide (Nissen‐Meyer et al ., ). These two peptides interact upon exposure to membrane‐like entities (Hildeng‐Hauge et al ., ) and appear to form a membrane‐penetrating helix–helix structure stabilized by helix–helix‐interacting GxxxG‐motifs upon contact with sensitive bacteria (Oppegård et al ., 2008; 2010; Rogne et al ., ). This motif is found in all two‐peptide bacteriocins that have currently been sequenced (Nissen‐Meyer et al ., ), and it has been proposed that the two peptides of many, if not all, two‐peptide bacteriocins form a membrane‐penetrating helix–helix structure that interacts with an integrated membrane protein in sensitive bacteria.…”

Development of Class IIa Bacteriocins as Therapeutic Agents

“…Chemically synthesized peptides with incorporated d -amino acids may be similarly expected to render the peptide less susceptible to proteolytic cleavage. Analogues of class IIb bacteriocin lactococcin G were synthesized with the N- and C-terminal residues replaced with d -amino acids, which decreased their susceptibility to exopeptidases without much effect on activity [54]. However, the extent of incorporation of d -amino acids has limitations.…”