CLUSTAL 2.1 multiple amino acid sequence alignment of cobra venom cytotoxins.

<p><b>A.</b> Amino acid sequence alignment of cobra venom cytotoxins, with resolved crystal structures, include 2CDX from <i>Naja naja atra</i>, 1ZAD (CTI/Vc1, underlined): <i>Naja naja oxiana</i>, 1CDT: <i>Naja mossambica mossambica</i>, 1CRE from <i>Naja naja atra</i>, 4OM5 from <i>Naja naja atra</i>, 2CRT from <i>Naja naja atra</i>, 1CB9 (CTII/Vc5, underlined) from <i>Naja naja oxiana</i>, 1UG4 from <i>Naja naja atra</i>, 2CCX from <i>Naja mossambica mossambica</i>, 1CVO from <i>Naja naja atra</i>. Conserved Cys residues are highlighted in yellow. Basic and acidic residues are highlighted in red and blue respectively. The amino acid residues located within each of the three loops (I-III) are highlighted in gray below the amino acid sequences. The variable residues Ser28 and Pro30 in loop 2, characteristic of S and P-type cytotoxins respectively, are highlighted in green. The hydrophobic residues of the three loops are bolded. The overall charge and isoelectric point (PI) value for each cytotoxin are shown in two columns to the right of the amino acid alignment. <b>B.</b> Ribbon diagrams of the crystal structures of CTI (PDB#1ZAD) and CTII (PDB#1CB9). All basic amino acid residues are represented as stick representations and labeled accordingly (Lys: lysine, Arg: arginine).</p

Cell permeabilizing activities of CTI and CTII on lipid bilayers.

<p>¹H-NMR spectra derived from the N⁺(CH₃)₃ groups of PC in large unilamellar liposomes composed of PC+ 10 mol% CL (<b>A</b> and <b>B</b>) or in PC+ 10 mol% PS (<b>C</b> and <b>D</b>) in the presence of potassium ferricyanide at 18°C and in the presence of CTII (<b>A</b> and <b>C,</b> top spectra) or of CTI (<b>B</b> and <b>D,</b> top spectra) at a cytotoxin/lipid molar ratio of 0.02. This figure shows a representative ¹H-NMR traces from three independent experiments that showed similar results. Each sample (<b>A-D</b>) was measured in triplicate.</p

Effects of cobra venom cytotoxins CTII and CTI on isolated mitochondria and large unilamellar liposomes.

<p>³¹P-NMR spectra of mitochondria or large unilamellar liposomes with different lipid compositions were monitored at 10°C. (<b>A</b>) ³¹P-NMR spectrum of a mitochondrial sample. (<b>B)</b>³¹P-NMR spectrum of mitochondria treated with 9 × 10⁻⁴ M of CTII. (<b>C</b>) ³¹P-NMR spectrum of mitochondria treated with 9 × 10⁻⁴ M of CTI. Broken lines in A and B are saturation spectra observed after applying a DANTE train of saturation pulses at the high-field side of the lamellar spectrum (see arrow with letter <b>S</b>). ³¹P-NMR spectra of large unilamellar liposomes of PC+ 10 mol% CL (<b>D</b>) and of PC+ 10 mol% PS (<b>E</b>) monitored at 18°C and treated with the indicated cytotoxin-lipid ratios of CTII and CTI. All ³¹P-NMR data shown in this figure from isolated mitochondria and large unilamellar liposomes is representative of two independent experiments that showed similar results. Each sample was measured in triplicate readings. <b>F-H.</b>³¹P-NMR spectra after applying a DANTE train of saturation pulses at the high-field peak of the lamellar spectrum of large unilamellar liposomes of PC+10 mol% CL treated with CTII (<b>F</b>) and CTI (<b>G</b>) and of PC+10 mol% PS treated with CTII (<b>H</b>) at the cytotoxin/lipid molar ratios of 0.01 (bottom spectra) and 0.02 (top spectra). Position of the signals in saturation spectra in <b>F</b>-<b>H</b> coincides with the position of ³¹P-NMR signal B.</p

Fusogenic effects of CTI and CTII on lipid bilayers.

<p>Calorimetric curves of large unilamellar liposomes containing: DMPC alone (curves 1 and 13); DPPC alone (curves 2 and 14); DMPC+ 5 mol% CL (curve 3); DPPC+ 5 mol% CL (curve 4); DMPC + 5mol% CL + CTII (curve 5); DPPC + 5 mol% CL + CTII (curve 6); DMPC + 5 mol% CL + CTI (curve 7); DPPC +5mol% CL + CTI (curve 8); a mixture of DMPC + 5 mol% CL and DPPC + 5 mol% CL (curve 9); mixture 9 after sonication (curve 10); mixture 9 + CTII (curve 11); mixture 9 + CTI (curve 12); DMPC + 10 mol% DMPS (curve 15); DPPC + 10 mol% DPPS (curve 16); DMPC + 1 0mol% DMPS + CTII (curve 17); DPPC + 10 mol% DPPS + CTII (curve 18); DMPC + 10 mol% DMPS + CTI (curve 19); DPPC + 10 mol% DPPS + CTI (curve 20); a mixture of DMPC + 10 mol% DMPS and DPPC + 10 mol% DPPS (curve 21); mixture 21 after sonication (curve 22); mixture 21 + CTII (curve 23); mixture 21 + CTI (curve 24). The cytotoxin/lipid molar ratio was set at 0.02 for all experimental conditions. This figure shows representative calorimetric traces from three independent experiments that showed similar results. Each sample (curves 1–21) was measured in triplicate.</p

Visual summary of residues in cytotoxins that interact and do not interact with lipids.

<p>Ribbon diagrams of CTI and CTII depicting the charged amino residues (ball and stick representations) that are predicted to interact with phospholipid head groups CL (top two panels), with PS (middle panels) or with PC (bottom two panels) based on the top ranked docking conformations identified by AutoDock simulations. Interacting charged amino acid residues are marked in yellow symbols and numbers and represented in stick mode. Charged amino acid residues in CTI and CTII that do not interact with PC, PS or CL are depicted in sphere mode representations and marked in white symbols and numbers. For a complete list of interactive residues and type of bonds produced with chemical groups of lipids. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.s003" target="_blank">S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.s005" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.s006" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.s007" target="_blank">S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.s008" target="_blank">S6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.s009" target="_blank">S7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.s010" target="_blank">S8</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.s011" target="_blank">S9</a> Tables).</p

Effects of CTII and CTI on the orientation and organization of lipid bilayers.

<p>EPR spectra of 5-DSA in oriented multibilayer films of PC+10 mol% CL (<b>A, B</b>) or in multibilayer films of PC+10 mol% PS (<b>C, D</b>) containing 5-DSA: lipid molar ratio of 1:100 at 18°C. The magnetic field that is parallel or perpendicular to the bilayer normal is indicated by arrows pointed at the respective EPR spectral lines. Multibilayer films were treated with CTII (<b>A, C</b>) or with CTI (<b>B, D</b>) at the indicated cytotoxin to lipid molar ratios. The graph shown in <b>E</b> represents the means and standard errors of the B/C ratios of the EPR spectra compiled from three independent experiments involving PC+ CL or PC+ PS multibilayer films treated or untreated with the indicated cytotoxins (*:P<0.05 for PC + PS/CTII vs. PC + PS/CTI, #:p<0.05 for PC + CL/ CTII vs. PC +CL/CTI). The graph shown in <b>F</b> represents the means and standard errors of the S parameters of the EPR spectra obtained from three independent experiments involving PC+ CL or PC+ PS multibilayer films treated or untreated with the indicated cytotoxins (*:P<0.05 for PC + PS/CTII vs. PC + PS/CTI). Values for (<i>B/C</i>)₀ and S₀ represent mean B/C and S values from lipids without cytotoxins and values for (<i>B/C</i>)_I and S_I represent mean B/C and S values from lipids treated at various cytotoxin to lipid molar ratios.</p

Proposed model describing the interaction of CTI and CTII with cell and mitochondrial membranes.

<p>Cytotoxins interacting with cell plasma membranes induce a contact of dehydrated surfaces of affected cells, which may result in the formation of various types of non-bilayer structures as shown in Inset: <b>A</b>—Phospholipids at a dehydrated contact zone which are not bound to a cytotoxin form transient inverted micelles responsible for ³¹P-NMR signal A shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.g002" target="_blank">Fig 2</a> which freely exchange with a lamellar phase. The movement of these inverted micelles to the periphery of a dehydrated contact results in the formation of a trilamellar structure (<b>B</b>), an intermediate stage that precedes membrane fusion. <b>C</b> Phospholipids at a dehydrated contact which are bound to a cytotoxin may facilitate the formation of stable inverted micelle with a cytotoxin in its center. Such cytotoxin-immobilized phospholipids do not exchange with a lamellar phase within a saturation time of 0.5 s and are responsible for ³¹P-NMR signal B (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.g002" target="_blank">Fig 2</a>). Following membrane fusion of neighboring cells, such an inverted micelle settles in a single membrane (<b>D</b>). Due to the high surface curvature, an inverted micelle eventually undergoes a lamellar phase with the eventual release of cytotoxin into the cytosol (left side of model). In addition, due to the insertion of cytotoxins in a membrane to the depth of one monolayer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.ref057" target="_blank">57</a>], cytotoxins induce an asymmetric enlargement of the membrane monolayer surfaces [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.ref056" target="_blank">56</a>] which may trigger formation of toroidal like pores (<b>E</b> (54)) which may account for ³¹P-NMR signal A (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.g002" target="_blank">Fig 2</a>) and the increased membrane permeability (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129248#pone.0129248.g004" target="_blank">Fig 4</a>). Once in the cytosol, cytotoxin translocates to the mitochondrion (right side of model). Upon binding to select few CL molecules on the OMM surface, cytotoxins, sequestered by inverted micelles may translocate into the inter-membrane space where cytotoxins may further promote the fusion between the inner leaflet of an OMM and the outer leaflet of an IMM. Such molecular events are predicted to disrupt mitochondrial integrity. Phospholipid head groups of CLs are colored yellow whereas phospholipid head groups of PCs are colored blue. The hydrophobic region of CTI (green oval) and CTII (red oval) is depicted with slanted lines.</p