Abstract
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.
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 physiology and pharmacology of pardaxins is rather complex; their effects range from interference with ionic transport in both the epithelium and nerve cells to morphological changes in the synaptic vesicles of lipid membranes (4 – 6)
Several biophysical studies show that the known sequences of pardaxins (Fig. 1) contain a single polypeptide chain with a high propensity to aggregate in aqueous solutions [7, 11]
Summary
The atomic coordinates and structure factors (code 1XC0) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). Pardaxins belong to a class of small amphipathic peptides that form part of the defense mechanism secreted by sole fish of the genus Pardachirus [1]. These polypeptides are postulated to be shark-repelling and toxic to several different organisms [2, 3]. The widely accepted mechanism for pardaxin interactions with these membranes is the so-called “barrel-stave” model This is a multistep mechanism in which the peptides are thought to a) bind the membrane in an ␣-helical structure, b) self-aggregate on the membrane surface, c) insert themselves into the hydrocarbon core of the membrane, and d) recruit more monomers, progressively increasing the size of the pore.
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