Solution structure of acidocin B, a circular bacteriocin produced by Lactobacillus acidophilus M46

Abstract

Acidocin B, a bacteriocin produced by Lactobacillus acidophilus M46, was originally reported to be a linear peptide composed of 59 amino acid residues. However, its high sequence similarity to gassericin A, a circular bacteriocin from Lactobacillus gasseri LA39, suggested that acidocin B might be circular as well. Acidocin B was purified from culture supernatant by a series of hydrophobic interaction chromatographic steps. Its circular nature was ascertained by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and tandem mass spectrometry (MS/MS) sequencing. The peptide sequence was found to consist of 58 amino acids with a molecular mass of 5,621.5 Da. The sequence of the acidocin B biosynthetic gene cluster was also determined and showed high nucleotide sequence similarity to that of gassericin A. The nuclear magnetic resonance (NMR) solution structure of acidocin B in sodium dodecyl sulfate micelles was elucidated, revealing that it is composed of four α-helices of similar length that are folded to form a compact, globular bundle with a central pore. This is a three-dimensional structure for a member of subgroup II circular bacteriocins, which are classified based on their isoelectric points of ∼7 or lower. Comparison of acidocin B with carnocyclin A, a subgroup I circular bacteriocin with four α-helices and a pI of 10, revealed differences in the overall folding. The observed variations could be attributed to inherent diversity in their physical properties, which also required the use of different solvent systems for three-dimensional structural elucidation.

FIG 1

(A) Sequence alignment of subgroup II circular bacteriocins using Clustal W (36). Conserved, conservative, and semiconservative substitutions are indicated by asterisks, colons, and semicolons, respectively. The previously proposed N-terminal amino acid for acidocin B is in brackets (22). (B) Schematic representation of the acidocin B gene cluster. The solid arrow indicates the structural bacteriocin gene aciA; the hatched arrow indicates aciT, encoding an ATP-binding protein; open arrows indicate aciB, aciC, aciD, aciI, and aciE, encoding proteins containing putative transmembrane domains. (C) Acidocin B precursor peptide. The cleavage site during maturation is indicated.

FIG 2

MALDI-TOF mass spectrum of acidocin B showing singly and doubly charged species consistent with an average molecular mass of 5,621.5 Da.

FIG 3

CD profile of acidocin B in SDS (solid line) and DPC (dashed line) micelles. The peptide exhibited similar α-helical contents in both micelles.

FIG 4

NMR solution structure of acidocin B (PDB code 2MWR): helix 1 is in blue, helix 2 is in purple, helix 3 is in cyan, and helix 4 is in orange. The arrow indicates the linkage of the N and C termini.

FIG 5

(A) Solution structure of acidocin B showing the amphipathicity of the helices. Hydrophobic residues are in green, while hydrophilic residues are in white. The arrow indicates the linkage of the N and C termini. (B) Hydrophobic surface map as generated from PyMOL (34). (C) Electrostatic potential surface map calculated using the APBS functionality of the PDB2PQR (version 1.8) online pipeline (35). Cationic regions are shown in red, while anionic regions are in blue.

FIG 6

Predicted structures of gassericin A and butyrivibriocin AR10 derived from homology modeling (SWISS-MODEL) (37) using the structure of acidocin B as the template. Basic residues are shown in red, and acidic residues are in blue. The arrow indicates the N- to C-terminal linkage.

FIG 7

Multiple-sequence alignment of known and putative (indicated by source organisms) subgroup II circular bacteriocin precursors using Clustal W (36). The known circular bacteriocins are acidocin B, gassericin A, and butyrivibriocin AR10. The UniProt accession numbers are in parentheses. The region corresponding to leader peptide sequences is underlined, and the highly conserved asparaginyl cleavage site is marked by an asterisk. Conserved residues (similarity threshold of 80%) are highlighted in black.