Defining the structure and receptor binding domain of the leaderless bacteriocin LsbB

Abstract

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 α-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.

Keywords: Antimicrobial Peptide (AMP); Bacteriocin Receptor; Leaderless Bacteriocin; Nuclear Magnetic Resonance (NMR); Peptide Interaction; Receptor; Structural Biology; Zn-dependent Protease.

FIGURE 1.

The NMR data obtained of LsbB in TFE (A and C) and DPC (B and D). The restraints used in structure calculation are shown in A and B. The signs * and § indicate that TALOS determined phi and psi torsion angle restraints for this residue were in the α-helical and β-sheet region of the Ramachandran plot, respectively. The lines indicate typical α-helical restraints obtained from analysis of the NOE spectra. The thicknesses of the lines shown are related to NOE cross peak intensities. CSI of C-α carbons in TFE and DPC is shown in C and D.

FIGURE 2.

LsbB NMR structures in TFE (A and C) and DPC (B and D). The structures ensembles of the 20 lowest energy structures superimposed over residues 3–17 are shown in A and B, and cartoon representations of the lowest energy structures are shown in C and D.

FIGURE 3.

Antimicrobial activity of LsbB (WT) and the LsbB mutants: Y23A, P24A, W25A, E26A, R27A, and K29A. Transformants of these mutants were spotted on a lawn of the indicator L. lactis Il1403. Inhibition is seen in the clear zones around the spotted cells.

FIGURE 4.

Sequence alignment of LsbB-like bacteriocins. Asterisks (*) indicate identical amino acids; Colons (:) indicate very similar amino acids, and dots (.) indicate similar amino acids.