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

Background: The bacteriocin LsbB targets a membrane-bound zinc-dependent peptidase. Results: The structure of LsbB was resolved by NMR. The C-terminal unstructured domains of LsbB and several other related bacteriocins were responsible for receptor binding. Conclusion: A subgroup of leaderless bacteriocins has been found to share a similar mechanism in receptor recognition. Significance: The study highlights the structure-function relationship of LsbB. LsbB is a class II leaderless lactococcal bacteriocin of 30 amino acids. In the present work, the structure and function relationship of LsbB was assessed. Structure determination by NMR spectroscopy showed that LsbB has an N-terminal -helix, whereas the C-terminal of the molecule remains unstructured. To define the receptor binding domain of LsbB, a competition assay was performed in which a systematic collection of truncated peptides of various lengths covering different parts of LsbB was used to inhibit the antimicrobial activity of LsbB. The results indicate that the outmost eight-amino acid sequence at the C-terminal end is likely to contain the receptor binding domain because only truncated fragments from this region could antagonize the antimicrobial activity of LsbB. Furthermore, alanine substitution revealed that the tryptophan in position 25 (Trp(25)) is crucial for the blocking activity of the truncated peptides, as well as for the antimicrobial activity of the full-length bacteriocin. LsbB shares significant sequence homology with five other leaderless bacteriocins, especially at their C-terminal halves where all contain a conserved KXXXGXXPWE motif, suggesting that they might recognize the same receptor as LsbB. This notion was supported by the fact that truncated peptides with sequences derived from the C-terminal regions of two LsbB-related bacteriocins inhibited the activity of LsbB, in the same manner as found with the truncated version of LsbB. Taken together, these structure-function studies provide strong evidence that the receptor-binding parts of LsbB and sequence-related bacteriocins are located in their C-terminal halves

Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death

Whole-genome sequencing of mutants with increased resistance against the two-peptide bacteriocin plantaricin JK reveals a putative receptor and potential docking site.

By whole-genome sequencing of resistant mutants, a putative receptor for plantaricin JK, a two-peptide bacteriocin produced by some Lactobacillus plantarum strains, was identified in Lactobacillus plantarum NCFB 965 and Weissella viridescens NCFB 1655. The receptors of the two species had 66% identical amino acid sequences and belong to the amino acid-polyamine-organocation (APC) transporter protein family. The resistant mutants contained point mutations in the protein-encoding gene resulting in either premature stop codons, leading to truncated versions of the protein, or single amino acid substitutions. The secondary structure of the W. viridescens protein was predicted to contain 12 transmembrane (TM) helices, a core structure shared by most members of the APC protein family. The single amino acid substitutions that resulted in resistant strains were located in a confined region of the protein that consists of TM helix 10, which is predicted to be part of an inner membrane pore, and an extracellular loop between TM helix 11 and 12. By use of template-based modeling a 3D structure model of the protein was obtained, which visualizes this mutational hotspot region and further strengthen the hypothesis that it represents a docking site for plantaricin JK