Structure and Orientation of Pardaxin Determined by NMR Experiments in Model Membranes*□S

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 H solution NMR spectroscopy. The peptide adopts a bend-helix-bend-helix motif with an angle between the two structure helices of 122 9°, 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 solventexposed. These results are complemented by solid-state NMR experiments on Pa4 in lipid bilayers. In particular, C-N rotational echo double-resonance experiments in multilamellar vesicles support the helical conformation of the C-terminal segment, whereas 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. 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 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). At minimum inhibitory concentrations (3 to 40 M), pardaxins are able to kill bacteria, whereas at higher concentrations ( 50 M), they lyse red blood cell membranes. In addition, pardaxins can disrupt the ionic transport of the osmoregulatory epithelium and presynaptic activity in mammals by forming voltage-dependent and ion-selective channels (1, 7, 8). An important characteristic of these membrane active peptides is their selective interaction with specific lipid membranes. Several mechanistic studies carried out with synthetic lipids suggest that pardaxins interact with the lipid surface by aggregating and forming pores, and eventually causing leakage of the cellular content (4). 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. Helicity, hydrophobic moment, hydrophobicity, charges, and the angle subtended by the hydrophilic/hydrophobic helix surfaces are all crucial structural parameters that modulate both the activity and selectivity of these membrane active peptides (9, 10). 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). It has been predicted that this family of peptides is composed of two -helices (from residues 2–10 and 13–27, respectively) that are joined by a short hinge flanked by two prolines at positions 7 and 13. The N-terminal segment of the peptide is thought to be inserted into the hydrophobic core of the lipid membranes, whereas the C-terminal helix probably represents the ion channel-lining segment of pardaxin (1, 12). Several CD studies have also been carried out on pardaxins. The conclusions of these studies are that pardaxins are generally unstructured in aqueous solutions and become highly helical upon the addition of increasing amounts of organic solvent (such as TFE) or synthetic lipids. In 1991, Zagorski and co-workers * This research was partly supported by an National Science Foundation Grant (CAREER development award to A. R) and funds from the National Institutes of Health (Grant AI054515-01A1). NMR instrumentation was provided with funds from the National Science Foundation (Grant BIR-961477) and the University of Minnesota Medical School. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □S The on-line version of this article (available at http://www.jbc.org) contains a supplemental table. 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/). ** To whom correspondence may be addressed: Biophysics Research Division and Dept. of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055. Tel.: 734-647-6572, Fax: 734-615-3790; E-mail: [email protected]. ‡‡ To whom correspondence may be addressed: Dept. of Chemistry, University of Minnesota, 139 Smith Hall, 207 Pleasant St. S.E., Minneapolis, MN 55455. Tel.: 612-625-0758; Fax 612-626-7541; E-mail: [email protected]. 1 The abbreviations used are: TFE, trifluoroethanol; DPC, sodium dodecylphosphocholine; MLV, multilamellar vesicle; REDOR, rotational echo double resonance; 2D, two dimensional; NOESY, nuclear Overhauser effect (or enhancement) spectroscopy; NOE, nuclear Overhauser effect; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 44, Issue of October 29, pp. 45815–45823, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.