Binding of tachyplesin I to DNA revealed by footprinting analysis: significant contribution of secondary structure to DNA binding and implication for biological action

Abstract

In view of the cationic amphipathic structure of tachyplesin I and antiparallel beta-sheet as a general DNA binding motif, DNA binding of the antimicrobial peptide has been examined. Several footprinting-like techniques using DNase I protection, dimethyl sulfate protection, and bleomycin- (BLM-) induced DNA cleavage were applied in this study. Some distinct footprints with DNase I are detected, and also the sequence-specific cleavage mode of the BLM-Fe(II) complex clearly is altered in the presence of tachyplesin I. In addition, methylation of the N-7 residue of guanine situated in the DNA major groove is not entirely inhibited (or activated) by tachyplesin I. The results suggest that tachyplesin I interacts with the minor groove of DNA duplex. Disappearance of the footprints by dithiothreitol-treated tachyplesin I and Ala-tachyplesin strongly suggests a significant contribution of secondary structure containing an antiparallel beta-sheet to the DNA binding of tachyplesin I. This is the first report on DNA interaction with a small peptide which contains a unique antiparallel beta-sheet structure. The mechanism for antimicrobial action of tachyplesin I has also been inferred.

Structure-Function Analysis of Tritrypticin, an Antibacterial Peptide of Innate Immune Origin

“…The nonlytic PR-39 kills bacteria by interrupting both DNA and protein synthesis (39). The DNA binding property of tachyplesin I has also been implicated in the antibiotic activity (40). The observed differences in the mechanism for bacterial killing appear to be consistent with the structural diversity among these molecules.…”

Functional Interrelationships between Cell Membrane and Cell Wall in Antimicrobial Peptide-Mediated Killing of Staphylococcus aureus

“…Thus, AP-induced perturbation of the CM, either by defined membrane poration or diffuse membrane permeabilization, has been felt to be required and sufficient to induce eventual microbial killing (25,43,49). However, recent investigations have also implicated intracellular targets and the autolytic enzyme system as participants in AP-induced lethal pathways (36,39,44,45,51). Moreover, several recent studies have documented that the composition of the CM or CW, as well as the overall microbial surface charge of certain pathogens (e.g., S. aureus), significantly influences the net antimicrobial activity of cationic APs (26,28,37,38).…”

Antimicrobial Peptides from Marine Invertebrates

“…The three tandem repeats of a tetrapeptide sequence, hydrophobic amino acid-Cys-hydrophobic amino acid-Arg, within the peptide indicate that its amphipathic nature, as confirmed by nuclear magnetic resonance structural investigations (36), is probably associated with biological activity (68). In view of the amphipathic cationic structure of tachyplesin and antiparallel ␤-sheets as a general DNA-binding motif, DNA binding using footprinting analysis of the peptide was examined and indicates that tachyplesin interacts with the minor groove of DNA duplex (96). Additional studies on tachyplesin found isopeptides, tachyplesin II and tachyplesin III, and also polyphemusins I and II in hemocytes of the American horseshoe crab Limulus polyphemus and the southeast Asian horseshoe crab Tachypleus gigas (Fig.…”