NMR structures and mutational analysis of the two peptides constituting the bacteriocin plantaricin S.

The structure of the individual peptides of the two-peptide bacteriocin plantaricin S, an antimicrobial peptide produced by a Lactobacillus plantarum strain, has been determined in DPC micelles. The two peptides of plantaricin S, Pls-α and Pls-ß, form an α-helix from and including residue 8 to 24 with a less structured region around residue 16-19 and an amphiphilic α-helix from and including residue 7 to 23, respectively. Activity assays on single amino acid-substituted GxxxG and GxxxG-like motifs show that substituting the Ser and Gly residues in the G9xxxG13 motif in Pls-α and the S17xxxG21 motif in Pls-ß reduced or drastically reduced the antimicrobial activity. The two-peptide bacteriocin muricidin contains GxxxG-like motifs at similar positions and displays 40-50% amino acid identity with plantaricin S. Activity assays of combinations of the peptides that constitute the bacteriocins plantaricin S and muricidin show that some combinations are highly active. Furthermore, sequence alignments show that the motifs important for plantaricin S activity align with identical motifs in muricidin. Based on sequence comparison and activity assays, a membrane-inserted model of plantaricin S in which the two peptides are oriented antiparallel relative to each other and where the GxxxG and GxxxG-like motifs important for activity come close in space, is proposed.

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.

Structure-Function Analysis of the Two-Peptide Bacteriocin Plantaricin EF.

Plantaricin EF is a two-peptide bacteriocin that depends on the complementary action of two different peptides (PlnE and PlnF) to function. The structures of the individual peptides have previously been analyzed by nuclear magnetic resonance spectroscopy ( Fimland, N. et al. ( 2008 ) , Biochim. Biophys. Acta 1784 , 1711 - 1719 ), but the bacteriocin structure and how the two peptides interact have not been determined. All two-peptide bacteriocins identified so far contain GxxxG motifs. These motifs, together with GxxxG-like motifs, are known to mediate helix-helix interactions in membrane proteins. We have mutated all GxxxG and GxxxG-like motifs in PlnE and PlnF in order to determine if any of these motifs are important for antimicrobial activity and thus possibly for interactions between PlnE and PlnF. Moreover, the aromatic amino acids Tyr and Trp in PlnE and PlnF were substituted, and four fusion polypeptides were constructed in order to investigate the relative orientation of PlnE and PlnF in target cell membranes. The results obtained with the fusion polypeptides indicate that PlnE and PlnF interact in an antiparallel manner and that the C-terminus of PlnE and N-terminus of PlnF are on the outer part of target cell membranes and the N-terminus of PlnE and C-terminus of PlnF are on the inner part. The preference for an aromatic residue at position 6 in PlnE suggests a positioning of this residue in or near the membrane interface on the cells inside. Mutations in the GxxxG motifs indicate that the G5xxxG9 motif in PlnE and the S26xxxG30 motif in PlnF are involved in helix-helix interactions. Atomistic molecular dynamics simulation of a structural model consistent with the results confirmed the stability of the structure and its orientation in membranes. The simulation approved the anticipated interactions and revealed additional interactions that further increase the stability of the proposed structure.

Interactions of a class IIb bacteriocin with a model lipid bilayer, investigated through molecular dynamics simulations.

The emergence of antibiotic resistant microorganisms poses an alarming threat to global health. Antimicrobial peptides (AMPs) are considered a possible effective alternative to conventional antibiotic therapies. An understanding of the mechanism of action of AMPs is needed in order to better control and optimize their bactericidal activity. Plantaricin EF is a heterodimeric AMP, consisting of two peptides Plantaricin E (PlnE) and Plantaricin F (PlnF). We studied the behavior of these peptides on the surface of a model lipid bilayer. We identified the residues that facilitate peptide-peptide interactions. We also identified residues that mediate interactions of the dimer with the membrane. PlnE interacts with the membrane through amino acids at both its termini, while only the N terminus of PlnF approaches the membrane. By comparing the activity of single-site mutants of the two-peptide bacteriocin and the simulations of the bacteriocin on the surface of a model lipid bilayer, structure activity relationships are proposed. These studies allow us to generate hypotheses that relate biophysical interactions observed in simulations with the experimentally measured activity. We find that single-site amino acid substitutions result in markedly stronger antimicrobial activity when they strengthen the interactions between the two peptides, while, concomitantly, they weaken peptide-membrane association. This effect is more pronounced in the case of the PlnE mutant (G20A), which interacts the strongest with PlnF and the weakest with the membrane while displaying the highest activity.

Structural basis and SAR for G007-LK, a lead stage 1,2,4-triazole based specific tankyrase 1/2 inhibitor.

Tankyrases 1 and 2 (TNKS1/2) are promising pharmacological biotargets with possible applications for the development of novel anticancer therapeutics. A focused structure-activity relationship study was conducted based on the tankyrase inhibitor JW74 (1). Chemical analoging of 1 improved the 1,2,4-triazole based core and led to 4-{5-[(E)-2-{4-(2-chlorophenyl)-5-[5-(methylsulfonyl)pyridin-2-yl]-4H-1,2,4-triazol-3-yl}ethenyl]-1,3,4-oxadiazol-2-yl}benzonitrile (G007-LK), a potent, "rule of 5" compliant and a metabolically stable TNKS1/2 inhibitor. G007-LK (66) displayed high selectivity toward tankyrases 1 and 2 with biochemical IC50 values of 46 nM and 25 nM, respectively, and a cellular IC50 value of 50 nM combined with an excellent pharmacokinetic profile in mice. The PARP domain of TNKS2 was cocrystallized with 66, and the X-ray structure was determined at 2.8 Å resolution in the space group P3221. The structure revealed that 66 binds to unique structural features in the extended adenosine binding pocket which forms the structural basis for the compound's high target selectivity and specificity. Our study provides a significantly optimized compound for targeting TNKS1/2 in vitro and in vivo.