Deletion of All Cysteines in Tachyplesin I Abolishes Hemolytic Activity and Retains Antimicrobial Activity and Lipopolysaccharide Selective Binding

Abstract

Tachyplesin I is a cyclic beta-sheet antimicrobial peptide isolated from the hemocytes of Tachypleus tridentatus. The four cysteine residues in tachyplesin I play a structural role in imparting amphipathicity to the peptide which has been shown to be essential for its activity. We investigated the role of amphipathicity using an analogue of tachyplesin I (TP-I), CDT (KWFRVYRGIYRRR-NH(2)), in which all four cysteines were deleted. Like TP-I, CDT shows antimicrobial activity and disrupts Escherichia coli outer membrane and model membranes mimicking bacterial inner membranes at micromolar concentrations. The CDT peptide does not cause hemolysis up to 200 microg/mL while TP-I showed about 10% hemolysis at 100 microg/mL and about 25% hemolysis at 150 microg/mL. Peptide-into-lipid titrations under isothermal conditions reveal that the interaction of CDT with lipid membranes is an enthalpy-driven process. Binding assays performed using fluorometry demonstrate that the peptide CDT binds and inserts into only negatively charged membranes. The peptide-induced thermotropic phase transition of MLVs formed of DMPC and the DMPC/DMPG (7:3) mixture suggests specific lipid-peptide interactions. The circular dichroism study shows that the peptide exists as an unordered structure in an aqueous buffer and adopts a more ordered beta-structure upon binding to negatively charged membrane. The NMR data suggest that CDT binding to negatively charged bilayers induces a change in the lipid headgroup conformation with the lipid headgroup moving out of the bilayer surface toward the water phase, and therefore, a barrel stave mechanism of membrane disruption is unlikely as the peptide is located near the headgroup region of lipids. The lamellar phase (31)P chemical shift spectra observed at various concentrations of the peptide in bilayers suggest that the peptide may function neither via fragmentation of bilayers nor by promoting nonlamellar structures. NMR and fluorescence data suggest that the presence of cholesterol inhibits the peptide binding to the bilayers. These properties help to explain that cysteine residues may not contribute to antimicrobial activity and that the loss of hemolytic activity is due to lack of hydrophobicity and amphipathicity.

NMR Structure of Pardaxin, a Pore-forming Antimicrobial Peptide, in Lipopolysaccharide Micelles

“…Current study identifies critical structural features of Pa4 for LPS recognition and provides a plausible mechanism of the outer membrane disruption. We believe that the LPS-bound structure of Pa4 and other finding in this study will be useful in the development of new antimicrobials with enhanced binding affinity for LPS (62,75).…”

The role of disulfide bridges in the 3-D structures of the antimicrobial peptides gomesin and protegrin-1: a molecular dynamics study

“…The impact of such a challenging problem has motivated numerous academic and pharmaceutical industry research groups to develop new drugs capable of dealing with the adaptation strategy (namely the selection of organisms adapted to some specific medium) that these organisms develop over time (Prates and Bloch Júnior, 2000;Fazio et al, 2006;Ramamoorthy et al, 2006;Che et al, 2008).…”

“…Our proposition is based on the study carried out with tachyplesin-I mutants, in which the four existing cysteines of the natural peptide were replaced by tyrosine residues (Rao, 1999;Ramamoorthy et al, 2006). In the study with tachyplesin-I, the authors found a great similarity between the native and the mutant conformations.…”

Comparative mode of action of novel hybrid peptide CS‐1a and its rearranged amphipathic analogue CS‐2a

“…For the lipid-binding experiment, different lipid concentrations of PC/PG SUVs (0, 85, 340, 680 and 1360 lM) or of PC SUVs (0, 83, 470, 748 and 1346 lM) were incubated with a fixed peptide concentration of 5 lM for 30 min as described previously [54]. Trp emission spectra were recorded using a 1-cm path length quartz cuvette on a spectrofluorometer (Jobin Yvon FluoroMax 3) with the excitation set at 280 nm using a slit width of 5 nm for both excitation and emission.…”