Abstract
The three-dimensional structure in dodecyl phosphocholine micelles of the 26-mer membrane-permeabilizing bacteriocin-like pheromone plantaricin A (PlnA) has been determined by use of nuclear magnetic resonance spectroscopy. The peptide was unstructured in water but became partly structured upon exposure to micelles. An amphiphilic alpha-helix stretching from residue 12 to 21 (possibly also including residues 22 and 23) was then formed in the C-terminal part of the peptide, whereas the N-terminal part remained largely unstructured. PlnA exerted its membrane-permeabilizing antimicrobial activity through a nonchiral interaction with the target cell membrane because the d-enantiomeric form had the same activity as the natural l-form. This nonchiral interaction involved the amphiphilic alpha-helical region in the C-terminal half of PlnA because a 17-mer fragment that contains the amphiphilic alpha-helical part of the peptide had antimicrobial potency that was similar to that of the l- and d-enantiomeric forms of PlnA. Also the pheromone activity of PlnA depended on this nonchiral interaction because both the l- and d-enantiomeric forms of the 17-mer fragment inhibited the pheromone activity. The pheromone activity also involved, however, a chiral interaction between the N-terminal part of PlnA and its receptor because high concentrations of the l-form (but not the d-form) of a 5-mer fragment derived from the N-terminal part of PlnA had pheromone activity. The results thus reveal a novel mechanism whereby peptide pheromones such as PlnA may function. An initial nonchiral interaction with membrane lipids induces alpha-helical structuring in a segment of the peptide pheromone. The peptide becomes thereby sufficiently structured and properly positioned in the membrane interface, thus enabling it to engage in a chiral interaction with its receptor in or near the membrane water interface. This membrane-interacting mode of action explains why some peptide pheromones/hormones such as PlnA sometimes display antimicrobial activity in addition to their pheromone activity.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
The results of this structure-function analysis indicate that some peptide pheromones/hormones such as plantaricin A (PlnA) function through a novel membrane-interacting mechanism whereby they adopt a membrane-induced ␣-helical structure that subsequently enables the peptides to interact with their receptors
The results indicate that the PlnA action as a pheromone involves an initial nonchiral interaction with membrane lipids, which induces ␣-helical structuring of the C-terminal part of the peptide (Figs. 3 and 4)
Summary
Purification, and Analysis of Peptides—The natural Lenantiomeric forms of PlnA-26 and -22 (PlnA-26-L and PlnA-22-L), the D-enantiomeric form of PlnA-22 (PlnA22-D), the 5-mer N-terminal fragment of PlnA-22-L (N-5-L), and the 17-mer C-terminal fragment of PlnA-22-L (PlnA-17-L) were all synthesized according to the sequence reported previously (Fig. 1) [5, 7]. Assay for Pheromone Activity—The pheromone activity was determined essentially as described earlier [7], using L. plantarum C11 cells with a bacteriocin-negative phenotype [6]. The transformation of cells from the bacteriocin-negative to the bacteriocin-producing phenotype upon adding peptides was determined by measuring the amount of bacteriocin produced after 6 h, using the assay for antimicrobial activity described above and the indicator strain L. plantarum 965. Restraints and Structure Calculation—Dihedral angle restraints were obtained by using the TALOS program [21] and the chemical shifts assigned by the application of the HSQC [19] and TOCSY. From this program we obtained restrictions for the torsion angles of the molecule from residue 12 to residue 21. The structures were visualized with the MOLMOL program [24]
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