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

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°, 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, 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 (Ͼ50 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.

Mapping the interaction surface of a membrane protein: Unveiling the conformational switch of phospholamban in calcium pump regulation

“…The boundaries of structural biology have expanded with the application of solution NMR to determine the structures of small membrane-bound peptides as well as large, multidomain membrane proteins (33)(34)(35)(36)(37)(38)(39). Here, we report the functional interaction between two membrane proteins probed by solution NMR in detergent micelles.…”

Membrane structure and conformational changes of the antibiotic heterodimeric peptide distinctin by solid-state NMR spectroscopy

“…In addition, this type of supramolecular polypeptide-lipid assemblies can exhibit many different morphologies that are best described by phase diagrams (3,39) where lysis, wormhole-(40) and carpet-model (41), or regions where the bilayer is unaffected or even stabilized, correspond to different regions in the diagram. The ''phase boundaries'' depend on lipid composition, temperature, salt, pH, and other environmental parameters, and thereby reflect the variable environments encountered by the peptides when interacting with the membranes of different species (39,42).…”

Antimicrobial peptides: an overview of a promising class of therapeutics

“…In particular, Ramamoorthy et al used specific membrane model systems and nuclear magnetic resonance (NMR) to characterize structure and dynamics of several AMPs such as pardaxins [36][37][38], MSI-78 (Pexiganan) and MSI-594 synthetic magainin analogues [39,40], MSI-843 lipopeptide [41], granulysin [42], tachyplesin [43] and a cyclic peptide subtilosin A [44]. In particular, the studies on pardaxin [36,38] have shown the role of cholesterol on inhibiting the membrane disruption and the role of the peptide on the formation of cholesterol-rich domains.…”