Pardaxin P-2

Pardaxins are a class of ichthyotoxic peptides isolated from fish mucous glands. Pardaxins physically interact with cell membranes by forming pores or voltage-gated ion channels that disrupt cellular functions. Here we report the high-resolution structure of synthetic pardaxin Pa4 in sodium dodecylphosphocholine micelles, as determined by (1)H solution NMR spectroscopy. The peptide adopts a bend-helix-bend-helix motif with an angle between the two structure helices of 122 +/- 9 degrees , making this structure substantially different from the one previously determined in organic solvents. In addition, paramagnetic solution NMR experiments on Pa4 in micelles reveal that except for the C terminus, the peptide is not solvent-exposed. These results are complemented by solid-state NMR experiments on Pa4 in lipid bilayers. In particular, (13)C-(15)N rotational echo double-resonance experiments in multilamellar vesicles support the helical conformation of the C-terminal segment, whereas (2)H NMR experiments show that the peptide induces considerable disorder in both the head-groups and the hydrophobic core of the bilayers. These solid-state NMR studies indicate that the C-terminal helix has a transmembrane orientation in DMPC bilayers, whereas in POPC bilayers, this domain is heterogeneously oriented on the lipid surface and undergoes slow motion on the NMR time scale. These new data help explain how the non-covalent interactions of Pa4 with lipid membranes induce a stable secondary structure and provide an atomic view of the membrane insertion process of Pa4.

Pardaxin, a 33-amino-acid pore-forming polypeptide toxin isolated from the Red Sea Moses sole Pardachirus marmoratus, has a helix-hinge-helix structure. This is a common structural motif found both in antibacterial peptides that can act selectively on bacterial membranes (e.g., cecropin), and in cytotoxic peptides that can lyse both mammalian and bacterial cells (e.g., melittin). Herein we show that pardaxin possesses a high antibacterial activity with a significantly reduced hemolytic activity towards human red blood cells (hRBC), compared with melittin. Its potency is comparable to that of other known native antibacterial peptides such as magainin, cecropins and dermaseptins. To determine the structural features responsible for the selective hemolytic and antibacterial activities, and the structural requirements for a high antibacterial activity, 8 truncated and modified pardaxin analogues were synthesized and structurally and functionally characterized. Each peptide was synthesized with a free carboxylate or amino group (i.e., aminated form) at its C-terminus. The aminated form of pardaxin has both high hemolytic and antibacterial activity. A truncated analogue, with 11 amino acids removed from the C-terminal domain, had dramatically reduced hemolytic activity. However, the aminated form of this analogue was significantly more potent that pardaxin against most bacteria tested, suggesting that the C-terminal tail of pardaxin is responsible for non-selective activity against erythrocytes and bacteria. Furthermore, a positive charge added to its N-terminus significantly increased its antibacterial activity and abolished its low hemolytic activity. The 22-amino-acid C-terminal domain and the short 11-amino-acid N-terminal domain were, in their aminated forms, active only against gram-positive bacteria. Secondary-structure determination using circular dichroism spectroscopy revealed that all the aminated analogues had 25-80% more alpha-helical content in 40% CF3CH2OH/water than their non-aminated forms. Using model phospholipid membranes it was found that all the analogues that were less hemolytic but had retained antibacterial activity could permeate acidicly charged phospholipid vesicles better than zwitterionic phospholipid vesicles, a property characteristics of all the native antibacterial peptides tested so far (e.g., cecropins, magainins and dermaseptins). Pardaxin and its analogues therefore represent a new class of antibacterial peptides that can serve as a basis for the design of therapeutic agents. Furthermore, negative-staining electron microscopy revealed that total inhibition of bacterial growth was due to total lysis of the bacterial wall. Therefore, it might be more difficult for bacteria to develop resistance to such a destructive mechanism, compared with the more specific mechanisms of the currently used antibiotics.

The cytolytic activities and conformational properties of pardaxin (GFFALIPKIISSPLFKTLLSAVGSALSSSGEQE), a 33-residue linear peptide that exhibits unusual shark repellent and cytolytic activities, and its analogues have been examined in aqueous environment and trifluoroethanol (TFE) using CD spectroscopy. A peptide corresponding to the 1-26 segment and an analogue where P7 has been changed to A show greater hemolytic activity than pardaxin. While the peptide corresponding to the N-terminal 18-residue segment does not exhibit hemolytic activity, its analogue where P7 is replaced by A is hemolytic. The secondary structural propensities of the peptides were inferred by deconvolution of the experimental spectra into pure components. Pardaxin, its variant where proline at position 7 was replaced by alanine, and shorter peptides corresponding to N-terminal segments exist in multiple conformations in aqueous medium that are comprised of beta-turn, beta-sheet, and distorted helical structures. With increasing proportions of TFE, while helical conformation predominates in all the peptides, both distorted and the regular alpha-helices appear to be populated. Analysis of CD spectra by deconvolution methods appears to be a powerful tool for delineating multiple conformations in peptides, especially membrane-active peptides that encounter media of different polarity ranging from aqueous environment to one of low dielectric constant in the hydrophobic interior of membranes. Our study provides further insights into the structural requirements for the biological activity of pardaxin and related peptides.