Solution Structure of Carnobacteriocin B2 and Implications for Structure−Activity Relationships among Type IIa Bacteriocins from Lactic Acid Bacteria^,

Abstract

Carnobacteriocin B2 (CbnB2), a type IIa bacteriocin, is a 48 residue antimicrobial peptide from the lactic acid bacterium Carnobacterium pisicola LV17B. Type IIa bacteriocins have a conserved YGNGVXC sequence near the N-terminus and usually contain a disulfide bridge. CbnB2 seemed to be unique in that its two cysteines (Cys9 and Cys14) could be isolated as free thiols [Quadri et al. (1994) J. Biol. Chem. 26, 12204-12211]. To establish the structural consequences of the presence or absence of a disulfide bridge and to investigate if the YGNGVXC sequence is a receptor-binding motif [Fleury et al. (1996) J. Biol. Chem. 271, 14421-14429], the three-dimensional solution structure of CbnB2 was determined by two-dimensional (1)H nuclear magnetic resonance (NMR) techniques. Mass spectroscopic and thiol modification experiments on CbnB2 and on model peptides, in conjunction with activity measurements, were used to verify the redox status of CbnB2. The results show that CbnB2 readily forms a disulfide bond and that this peptide has full antimicrobial activity. NMR results indicate that CbnB2 in trifluoroethanol (TFE) has a well-defined central helical structure (residues 18-39) but a disordered N terminus. Comparison of the CbnB2 structure with the refined solution structure of leucocin A (LeuA), another type IIa bacteriocin, indicates that the central helical structure is conserved between the two peptides despite differences in sequence but that the N-terminal structure (a proposed receptor binding site) is not. This is unexpected because LeuA and CbnB2 exhibit >66% sequence identity in the first 24 residues. This suggests that the N-terminus, which had been proposed [Fleury et al. (1996) J. Biol. Chem. 271, 14421-14429] to be a receptor binding site of type IIa bacteriocins, may not be directly involved and that recognition of the amphiphilic helical portion is the critical feature.

Structure-Function Analysis of Immunity Proteins of Pediocin-Like Bacteriocins: C-Terminal Parts of Immunity Proteins Are Involved in Specific Recognition of Cognate Bacteriocins

“…Interestingly, the CD spectra revealed that there was no further increase in the structuring or ␣-helical content when EntA-im was exposed to dodecylphosphocholine micelles or DOPG liposomes (results not shown), also indicating that the immunity proteins, in contrast to the bacteriocins (17,33,38), do not interact extensively with membranes. This is consistent with their largely hydrophilic character and the finding that only a minor fraction (less than 1%) of the immunity protein for the pediocin-like bacteriocin carnobacteriocin B2 is associated with the cell membrane (27).…”

“…The C-terminal parts of these bacteriocins are important determinants of their target cell specificities (13). At least 20 pediocinlike bacteriocins have been characterized (12,22,26; see reference 15 for original references), and the three-dimensional structures of some of these have been analyzed by nuclear magnetic resonance spectroscopy and mutagenesis (14,15,17,33,38).…”

NMR solution structure of the precursor for carnobacteriocin B2, an antimicrobial peptide from Carnobacterium piscicola

“…However, this material displays CD spectra and antimicrobial properties indistinguishable from the complete preCbnB2. The two missing hydrophilic residues are at the C-terminus of the peptide, which is unstructured in the mature CbnB2 [9], and distant from the N-terminal leader portion. The C-terminal section is also random coil in preCbnB2(1-64) (see below).…”

“…As these bacteriocins are well known to interact with membrane lipids and recognize a chiral membrane-bound receptor molecule [2,11], the a-helical portion of the structure of mature type IIa bacteriocins is critical for their biological activity [8][9][10]. CD experiments (data not shown) demonstrate that, like the mature bacteriocin, preCbnB2 is unstructured in pure water but contains one or more a-helical sections in more lipophilic trifluoroethanol/water mixtures.…”

1.6-Å Crystal Structure of EntA-im

“…Moreover, they have very similar primary structures, especially in the N-terminal domain (6). NMR studies indicate that pediocin-like bacteriocins contain two structural units, a cationic N-terminal ␤-sheet domain and a hydrophobic C-terminal domain (7)(8)(9), which in most of these peptides appears to form a hairpin-like structure (7). These two domains are separated by a flexible hinge that enables the two domains to move relative to each other.…”