Impact of membrane partitioning on the spatial structure of an S-type cobra cytotoxin

<p>Cobra cytotoxins (CTs) belong to the three-fingered protein family. They are classified into S- and P-types, the latter exhibiting higher membrane-perturbing capacity. In this work, we investigated the interaction of CTs with phospholipid bilayers, using coarse-grained (CG) and full-atom (FA) molecular dynamics (MD). The object of this work is a CT of an S-type, cytotoxin I (CT1) from <i>N.oxiana</i> venom. Its spatial structure in aqueous solution and in the micelles of dodecylphosphocholine (DPC) were determined by ¹H-NMR spectroscopy. Then, via CG- and FA MD-computations, we evaluated partitioning of CT1 molecule into palmitoyloleoylphosphatidylcholine (POPC) membrane, using the toxin spatial models, obtained either in aqueous solution, or detergent micelle. The latter model exhibits minimal structural changes upon partitioning into the membrane, while the former deviates from the starting conformation, loosing the tightly bound water molecule in the loop-2. These data show that the structural changes elicited by CT1 molecule upon incorporation into DPC micelle take place likely in the lipid membrane, although the mode of the interaction of this toxin with DPC micelle (with the tips of the all three loops) is different from its mode in POPC membrane (primarily with the tip of the loop-1 and both the tips of the loop-1 and loop-2).</p

Inhibition experiments with PP PLA₂.

<p>(A) Dose-response curve of PP PLA₂ inhibitory action on the ACh-evoked (25 μM ACh) ionic currents mediated by human α9α10 nAChR heterologously expressed in <i>Xenopus</i> oocytes. (B) Inhibition by PP PLA₂ of the initial rate of specific [¹²⁵I]-α-Bgt binding to <i>T</i>. <i>californica</i> and human hα7 nAChRs expessed in GH₄C₁ cells. Only 30% of binding sites in hα7 nAChRs could be protected from [¹²⁵I]-α-Bgt binding. (C) Inhibition by PP PLA₂ of specific [¹²⁵I]-α-Bgt binding to ECD of human α9 nAChR. IC₅₀ 5.5 μM.</p

Interaction of non-venom proteins with <i>T</i>.<i>californica</i> nAChR.

<p>Inhibition of the initial rate of specific [¹²⁵I]-α-Bgt binding to <i>T</i>.<i>californica</i> nAChR by RNAse (squares) and cytochrome C (circles).</p

SPR recordings of Cro interaction with AChBP from <i>L</i>. <i>stanalis</i>.

<p>An arrow indicates injection of the analyte. Line 1 corresponds to injection of buffer solution. Curve 2–5 correspond to injections of solutions with Cro at concentrations of 5.5, 11.5, 23 and 46 μg/ml, respectively.</p

Interaction of Cro with nAChR of <i>T</i>. <i>californica</i> electric organ, human neuronal α7 nAChR and AChBP.

<p>(A) Inhibition of the initial rate of specific [¹²⁵I]-α-Bgt binding to <i>T</i>.<i>californica</i> nAChRs by Cro. Points were fit to a 2-site model with affinity for Cro of 30 nM and 260 nM. (B) Inhibition of the initial rate of specific [¹²⁵I]-α-Bgt binding to human α7 nAChRs by Cro. Two binding sites with affinity for Cro differing more than 3 orders of magnitude were revealed. (C) Inhibition of the initial rate of specific [¹²⁵I]-α-Bgt binding to acetylcholine-binding protein from <i>L</i>. <i>staganlis</i> by Cro.</p

Isolation of vurtoxin and Vur-S49 by reverse-phase HPLC.

<p>Separation was done on a Discovery BIO Wide Pore C18 column (10×250 mm, Supelco) in a gradient of 25–40% (v/v) acetonitrile in 60 min in the presence of 0.1% (v/v) trifluoroacetic acid, at a flow rate of 2.0 ml/min. Fraction containing Vur-S49 (4) and vurtoxin (11) are indicated by horizontal bars.</p

Comparison of CM-II and vurtoxin effects on Cyt- and ACh-evoked currents in neurons containing predominantly nAChR-Ls-2 or nAChR-Ls-1.

<p><i>A</i> - A neuron with low sensitivity to ImI against ACh-induced current (nAChR-Ls-2). CM-II at concentration of 500 nM heavily decreased the Cyt-elicited current but affected very slightly the response to ACh. <i>B</i> - Vurtoxin at concentration of 8 µM decreased the currents elicited by Cyt or ACh in two neurons predominantly containing nAChR-Ls2 (up line) or nAChR-Ls1 (middle line) to 0.56 and 0.60 of the controls, respectively. In the third neuron with predominant nAChR-Ls-2 (bottom line) vurtoxin at the same concentration reduced the ACh-evoked current only to 0.80.</p

Inhibition of acetylcholine or cytisine-elicited current in <i>L</i>.<i>stagnalis</i> neurons by PP PLA₂ and β-Bgt.

<p>(A) Representative recordings from a neuron in control, after a 5-min incubation with PP PLA₂ at three different concentrations, and after PP PLA₂ wash out. (B) Dependence of ACh- or cytisine-evoked current suppression on PP PLA₂ (n = 7) and β-Bgt (n = 4) concentration.</p

Determination of antagonism type for PP PLA₂.

<p>Dependence of cytisine (A) or acetylcholine (B) induced currents on agonist concentration in control (closed circles and triangles) and after 5 min treatment with PP PLA₂ at 3 μM (open symbols), (n = 5 and 1, respectively).</p

Affinities for inhibiting different targets and phospholipolytic activities of PLA₂s.

<p>* The data obtained for antagonizing action on currents induced by ACh in nAChR-Ls-1 neurons and by Cyt were combined</p><p>** Not determined</p><p>Affinities for inhibiting different targets and phospholipolytic activities of PLA₂s.</p