1-palmitoyl-2-oleoylphosphatidylethanolamine and 1-2-dioleoylphosphatidylserine

Cardiotoxins (CTXs) are single polypeptide chain consisting of 59-62 amino acids with four disulfide bridges and globular proteins of simple β-sheet folds. The CTXs are one of principal toxic components causing haemolysis and damaging various cells and belong to three-finger toxin (TFT) superfamily of snake venoms. However, there is no natural or synthetic small molecular inhibitor to the protein toxins to date. In the present study, modes of interaction of cardiotoxin 1 (CTX1) from Indian cobra (Naja naja) with heterogeneous erythrocyte membrane (EM) model system have been extensively examined by using all-atom molecular dynamics (MD) simulations in near physiological conditions and comprehensive analyses of the MD data revealed two distinct principal regions ('head groove' and 'loop groove') of the protein toxin for establishing structural interactions with the EM system. Moreover, combined analyses of data from high-throughput virtual screening of NCI small molecular database, in vitro haemolytic assays for top-hits of the chemical compounds against crude venom of Naja naja and as well CTXs purified from the venom and pharmacokinetic examinations on the chemical compounds retarding haemolytic activities of CTXs suggested that Etidronic acid and Zoledronic acid are promising prototypic chemical inhibitors to CTXs of snake venoms.

Topics: Amino Acid Sequence; Animals; Antidotes; Cholesterol; Cobra Cardiotoxin Proteins; Diphosphonates; Disulfides; Elapid Venoms; Elapidae; Erythrocyte Membrane; Etidronic Acid; Hemolysis; High-Throughput Screening Assays; Humans; Imidazoles; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Protein Domains; Protein Structure, Secondary; Small Molecule Libraries; Structure-Activity Relationship; User-Computer Interface; Zoledronic Acid

The SNAREs SNAP25 and SNAP23 are proteins that are initially cytosolic after translation, but then become stably attached to the cell membrane through palmitoylation of cysteine residues. For palmitoylation to occur, membrane association is a prerequisite, but it is unclear which motif may increase the affinities of the proteins for the target membrane. In experiments with rat neuroendocrine cells, we find that a few basic amino acids in the cysteine-rich region of SNAP25 and SNAP23 are essential for plasma membrane targeting. Reconstitution of membrane-protein binding in a liposome assay shows that the mechanism involves protein electrostatics between basic amino acid residues and acidic lipids such as phosphoinositides that play a primary role in these interactions. Hence, we identify an electrostatic anchoring mechanism underlying initial plasma membrane contact by SNARE proteins, which subsequently become palmitoylated at the plasma membrane.

Topics: Amino Acid Motifs; Animals; Binding Sites; Cell Membrane; Cloning, Molecular; Escherichia coli; Gene Expression; Liposomes; Lipoylation; PC12 Cells; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Plasmids; Protein Binding; Protein Processing, Post-Translational; Protein Transport; Rats; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Static Electricity; Synaptosomal-Associated Protein 25; Vesicular Transport Proteins

Antimicrobial peptides (AMPs) are an emerging class of antibiotics for controlling health effects of antibiotic-resistant microbial strains. Protegrin-1 (PG-1) is a model antibiotic among beta-sheet AMPs. Antibiotic activity of AMPs involves cell membrane damage, yet their membrane interactions, their 3D membrane-associated structures and the mechanism underlying their ability to disrupt cell membrane are poorly understood. Using complementary approaches, including molecular dynamics simulations, atomic force microscopy (AFM) imaging, and planar lipid bilayer reconstitution, we provide computational and experimental evidence that PG-1, a beta-hairpin peptide, forms ion channels. Simulations indicate that PG-1 forms channel-like structures with loosely attached subunits when reconstituted in anionic lipid bilayers. AFM images show the presence of channel-like structures when PG-1 is reconstituted in dioleoylphosphatidylserine/palmitoyloleoyl phosphatidylethanolamine bilayers or added to preformed bilayers. Planar lipid bilayer electrical recordings show multiple single channel conductances that are consistent with the heterogeneous oligomeric channel structures seen in AFM images. PG-1 channel formation seems to be lipid-dependent: PG-1 does not easily show ion channel electrical activity in phosphatidylcholine membranes, but readily shows channel activity in membranes rich in phosphatidylethanolamine or phosphatidylserine. The combined results support a model wherein the beta-hairpin PG-1 peptide acts as an antibiotic by altering cell ionic homeostasis through ion channel formation in cell membranes.

Topics: Animals; Antimicrobial Cationic Peptides; Electric Conductivity; Lipid Bilayers; Membrane Potentials; Microscopy, Atomic Force; Molecular Dynamics Simulation; Phosphatidylethanolamines; Phosphatidylserines; Probability; Protein Structure, Secondary; Swine

To investigate the effect of lipid structure upon the membrane topography of hydrophobic helices, the behavior of hydrophobic peptides was studied in model membrane vesicles. To define topography, fluorescence and fluorescence quenching methods were used to determine the location of a Trp at the center of the hydrophobic sequence. For peptides with cationic residues flanking the hydrophobic sequence, the stability of the transmembrane (TM) configuration (relative to a membrane-bound non-TM state) increased as a function of lipid composition on the order: 1:1 (mol:mol) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine approximately 6:4 POPC:cholesterol

Topics: Amino Acid Sequence; Anions; Cholesterol; Drug Stability; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Membrane Lipids; Membrane Proteins; Membranes, Artificial; Molecular Conformation; Molecular Sequence Data; Peptides; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylserines; Protein Structure, Secondary; Spectrometry, Fluorescence; Static Electricity; Tryptophan