Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics

<p>Abstract</p> <p>Background</p> <p>The snake venom group IIA secreted phospholipases A₂(SVPLA₂), present in the <it>Viperidae </it>snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA₂-inhibitors from snake serum; and (iii) coagulation factors present in human blood.</p> <p>Results</p> <p>Using surface plasmon resonance (SPR) protein-protein interaction measurements and an <it>in vitro </it>biological test of inhibition of prothrombinase activity, we identify a number of <it>Viperidae </it>venom SVPLA₂s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa) via a non-catalytic, PL-independent mechanism. We classify the SVPLA₂s in four groups, depending on the strength of their binding.</p> <p>Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA₂and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA₂s. This site is composed of the following regions: helices A and B, the Ca²⁺loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA₂binding site residues belong also to the interfacial binding site (IBS). The interface in FXa involves both, the light and heavy chains.</p> <p>Conclusion</p> <p>We have experimentally identified several strong FXa-binding SVPLA₂s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA₂s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors.</p

New insights into interactions between the nucleotide-binding domain of CFTR and keratin 8

The intermediate filament protein keratin 8 (K8) interacts with the nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508-CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for cystic fibrosis patients bearing the ΔF508 mutation. Here we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen–deuterium exchange coupled with mass spectrometry (HDX-MS) on recombinant wild-type (wt) NBD1 and ΔF508-NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt-NBD1 and ΔF508-NBD1. Finally, we performed HDX-MS analysis of the NBD1 molecules and full-length K8, revealing hydrogen-bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508-NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1. This article is protected by copyright. All rights reserved

Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis

Crystallographic characterization of functional sites of crotoxin and ammodytoxin, potent b-neurotoxins from Viperidae venom

International audiencea b s t r a c t This review will focus on a description of the three-dimensional structures of two b-neurotoxins, the monomeric PLA 2 ammodytoxin from Vipera ammodytes ammodytes, and heterodimeric crotoxin from Crotalus durissus terrificus, and a detailed structural analysis of their multiple functional sites. We have recently determined at high resolution the crystal structures of two natural isoforms of ammodytoxin (AtxA and AtxC) (Saul et al., 2010) which exhibit different toxicity profiles and different anticoagulant properties. Compara-tive structural analysis of these two PLA 2 isoforms, which differ only by two amino acid residues, allowed us to detect local conformational changes and delineate the role of critical residues in the anticoagulant and neurotoxic functions of these PLA 2 (Saul et al., 2010). We have also determined, at 1.35 Å resolution, the crystal structure of heterodimeric crotoxin (Faure et al., 2011). The three-dimensional structure of crotoxin revealed details of the binding interface between its acidic (CA) and basic (CB) subunits and allowed us to identify key residues involved in the stability and toxicity of this potent heterodimeric b-neurotoxin (Faure et al., 2011). The precise spatial orientation of the three covalently linked polypeptide chains in the mature CA subunit complexed with CB helps us to understand the role played by critical residues of the CA subunit in the increased toxicity of the cro-toxin complex. Since the CA subunit is a natural inhibitor of the catalytic and anticoagulant activities of CB, identification of the CA–CB binding interface describes residues involved in this inhibition. We propose future research directions based on knowledge of the recently reported 3D structures of crotoxin and ammodytoxin

Relations entre la fonction venimeuse et la fonction immunitaire innée

La fonction venimeuse est étudiée dans ses rapports avec la fonction immunitaire innée à partir de deux exemples choisis dans les venins de scorpions puis dans les venins de serpents. Dans le premier exemple, l’analyse des structures des toxines de scorpion et des défensines amène à considérer la réelle intrication des deux fonctions. Dans le second exemple, l’analyse de la fonction des inhibiteurs de la phospholipase A2 (PLA2) neurotoxique conduit à l’analyse de structures homologues du système immunitaire inné de vertébrés (mammifères) pour aboutir à une même conclusion. Les venins de scorpions contiennent principalement des toxines actives sur les canaux ioniques membranaires dont la structure est comparable à celle de leurs défensines circulantes. Fonctionnellement, il n’y a pas d’erreur : les défensines se trouvent dans l’hémolymphe des scorpions, les toxines dans le venin utilisé dans l’immobilisation des proies. Toxines et défensines possèdent une homologie structurale et contiennent un motif tridimensionnel CSαβ conservé dit aussi « Cysteine-Stabilized alpha-helical and beta-sheet fold », composé de ponts disulfures conservés qui unissent une hélice α et des feuillets β antiparallèles. Une analyse génomique a permis aux auteurs de conclure que les toxines longues du venin de scorpion se sont précocement isolées d’un ensemble ayant donné naissance d’une part à l’ensemble « toxines longues » du venin et d’autre part à l’ensemble « toxines courtes et défensines ». Il y aurait donc bien un ancêtre commun aux défensines et aux toxines du venin. Une autre équipe a pu synthétiser une toxine courte de venin de scorpion à partir d’une défensine d’un hyménoptère parasitoïde. Enfin, un peptide antibactérien de scorpion, l’androctonine, dont la structure est comparable à celle d’une toxine de venin de cône, a montré que les deux molécules possèdent la même haute affinité pour le récepteur post-synaptique à l’acétylcholine de Torpedo sp. Les inhibiteurs naturels de phospholipases A2, identifiés principalement dans le sang de serpents et plus rarement dans certaines glandes à venin, expliquent la résistance habituelle des serpents venimeux vis-à-vis de leur propre venin, comme s’ils étaient protégés naturellement contre l’une des catégories de toxines les plus puissantes de leur venin. Les inhibiteurs de PLA2 ou PLI possèdent tous des structures variées qui les apparentent aux protéines de l’immunité innée, comme les lectines et les récepteurs Toll notamment, ce qui les différencie nettement des anticorps neutralisants. On trouve donc, chez des vertébrés comme chez des invertébrés, des molécules neutralisant les toxines qui sont composées de molécules apparentées aux protéines de l’immunité innée. Au total, ni dans les structures ni dans le mode de fonctionnement ni dans la phylogénie, la fonction venimeuse ne se détache véritablement de la fonction immunitaire innée

Crystal Structure of Isoform CBd of the Basic Phospholipase A2 Subunit of Crotoxin: Description of the Structural Framework of CB for Interaction with Protein Targets

International audienceCrotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic β-neurotoxin that exists in individual snake venom as a mixture of isoforms of a basic phospholipase A2 (PLA2) subunit (CBa2, CBb, CBc, and CBd) and acidic subunit (CA1–4). Specific natural mutations in CB isoforms are implicated in functional differences between crotoxin isoforms. The three-dimensional structure of two individual CB isoforms (CBa2, CBc), and one isoform in a crotoxin (CA2CBb) complex, have been previously reported. This study concerns CBd, which by interaction with various protein targets exhibits many physiological or pharmacological functions. It binds with high affinity to presynaptic receptors showing neurotoxicity, but also interacts with human coagulation factor Xa (hFXa), exhibiting anticoagulant effect, and acts as a positive allosteric modulator and corrector of mutated chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), implicated in cystic fibrosis. Thus, CBd represents a novel family of agents that have potential in identifying new drug leads related to anticoagulant and anti-cystic fibrosis function. We determined here the X-ray structure of CBd and compare it with the three other natural isoforms of CB. The structural role of specific amino acid variations between CB isoforms are analyzed and the structural framework of CB for interaction with protein targets is described

Actin Dynamics Is Controlled by a Casein Kinase II and Phosphatase 2C Interplay on Toxoplasma gondii Toxofilin

Actin polymerization in Apicomplexa protozoa is central to parasite motility and host cell invasion. Toxofilin has been characterized as a protein that sequesters actin monomers and caps actin filaments in Toxoplasma gondii. Herein, we show that Toxofilin properties in vivo as in vitro depend on its phosphorylation. We identify a novel parasitic type 2C phosphatase that binds the Toxofilin/G-actin complex and a casein kinase II-like activity in the cytosol, both of which modulate the phosphorylation status of Toxofilin serine(53). The interplay of these two molecules controls Toxofilin binding of G-actin as well as actin dynamics in vivo. Such functional interactions should play a major role in actin sequestration, a central feature of actin dynamics in Apicomplexa that underlies the spectacular speed and nature of parasite gliding motility

Toxins: New Targets and New Functions

International audienc

Characterization of a human coagulation factor Xa-binding site on snake venom phospholipases Aby affinity binding studies and molecular bioinformatics-5

<p><b>Copyright information:</b></p><p>Taken from "Characterization of a human coagulation factor Xa-binding site on snake venom phospholipases Aby affinity binding studies and molecular bioinformatics"</p><p>http://www.biomedcentral.com/1472-6807/7/82</p><p>BMC Structural Biology 2007;7():82-82.</p><p>Published online 6 Dec 2007</p><p>PMCID:PMC2248580.</p><p></p