{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T06:23:20Z","timestamp":1788589400118,"version":"build-2803163510"},"reference-count":51,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2020,11,13]],"date-time":"2020-11-13T00:00:00Z","timestamp":1605225600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic \u03b2-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\u20134). 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.<\/jats:p>","DOI":"10.3390\/molecules25225290","type":"journal-article","created":{"date-parts":[[2020,11,13]],"date-time":"2020-11-13T10:32:47Z","timestamp":1605263567000},"page":"5290","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["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"],"prefix":"10.3390","volume":"25","author":[{"given":"Dorota","family":"Nemecz","sequence":"first","affiliation":[{"name":"Institut Pasteur, R\u00e9cepteurs-Canaux, CNRS UMR 3571, D\u00e9partement de Neuroscience, 25, rue du Dr. Roux, F-75015 Paris, France"},{"name":"Biochemistry Department, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University, 87-100 Torun, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maciej","family":"Ostrowski","sequence":"additional","affiliation":[{"name":"Institut Pasteur, R\u00e9cepteurs-Canaux, CNRS UMR 3571, D\u00e9partement de Neuroscience, 25, rue du Dr. Roux, F-75015 Paris, France"},{"name":"Biochemistry Department, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University, 87-100 Torun, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marc","family":"Ravatin","sequence":"additional","affiliation":[{"name":"Institut Pasteur, R\u00e9cepteurs-Canaux, CNRS UMR 3571, D\u00e9partement de Neuroscience, 25, rue du Dr. Roux, F-75015 Paris, France"},{"name":"Sanofi R&amp;D, Integrated Drug Discovery-High Content Biology, 94400 Vitry-sur-Seine, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frederick","family":"Saul","sequence":"additional","affiliation":[{"name":"Institut Pasteur, Plateforme de Cristallographie-C2RT, CNRS UMR 3528, 75015 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6461-7558","authenticated-orcid":false,"given":"Grazyna","family":"Faure","sequence":"additional","affiliation":[{"name":"Institut Pasteur, R\u00e9cepteurs-Canaux, CNRS UMR 3571, D\u00e9partement de Neuroscience, 25, rue du Dr. Roux, F-75015 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,11,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1560","DOI":"10.1073\/pnas.68.7.1560","article-title":"Biological roles of the two components of crotoxin","volume":"68","author":"Hendon","year":"1971","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1016\/0041-0101(87)90246-7","article-title":"Several isoforms of crotoxin are present in individual venoms from the South American rattlesnake Crotalus durissus terrificus","volume":"25","author":"Faure","year":"1987","journal-title":"Toxicon"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1021\/bi00402a036","article-title":"Crotoxin, a phospholipase A2 neurotoxin from the South American rattlesnake crotalus durissus terrificus: Purification of several isoforms and comparison of their molecular structure and of their biological activities","volume":"27","author":"Faure","year":"1988","journal-title":"Biochemistry"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"8074","DOI":"10.1021\/bi00246a028","article-title":"Multiplicity of acidic subunit isoforms of crotoxin, the phospholipase A2 neurotoxin from Crotalus durissus terrificus venom, results from posttranslational modifications","volume":"30","author":"Faure","year":"1991","journal-title":"Biochemistry"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1111\/j.1432-1033.1993.tb17946.x","article-title":"Comparison of crotoxin isoforms reveals that stability of the complex plays a major role in its pharmacological action","volume":"214","author":"Faure","year":"1993","journal-title":"Eur. J. Biochem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1111\/j.1476-5381.1977.tb07553.x","article-title":"The mode of action at the mouse neuromuscular junction of the phospholipase A\u2014Crotapotin complex isolated from venom of the South American rattlesnake","volume":"61","author":"Hawgood","year":"1977","journal-title":"Br. J. Pharmacol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2779","DOI":"10.1021\/bi9612374","article-title":"Neurotoxic phospholipases A2 ammodytoxin and crotoxin bind to distinct high-affinity protein acceptors in Torpedo marmorata electric organ","volume":"36","author":"Krizaj","year":"1997","journal-title":"Biochemistry"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1016\/S0041-0101(02)00394-X","article-title":"Crotoxin acceptor protein isolated from Torpedo electric organ: Binding properties to crotoxin by surface plasmon resonance","volume":"41","author":"Faure","year":"2003","journal-title":"Toxicon"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Shimizu, J.F., Pereira, C.M., Bittar, C., Batista, M.N., Campos, G.R.F., da Silva, S., Cintra, A.C.O., Zothner, C., Harris, M., and Sampaio, S.V. (2017). Multiple effects of toxins isolated from Crotalus durissus terrificus on the hepatitis C virus life cycle. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0187857"},{"key":"ref_10","first-page":"1033","article-title":"Phase I and pharmacokinetics study of crotoxin (cytotoxic PLA2, NSC-624244) in patients with advanced cancer","volume":"8","author":"Cura","year":"2002","journal-title":"Clin. Cancer Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3009","DOI":"10.3892\/mmr.2014.2620","article-title":"Crotoxin suppresses the tumorigenic properties and enhances the antitumor activity of Iressa\u00ae (gefinitib) in human lung adenocarcinoma SPCA-1 cells","volume":"10","author":"Wang","year":"2014","journal-title":"Mol. Med. Rep."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"540","DOI":"10.1111\/j.1745-7254.2007.00530.x","article-title":"Autophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF-7 cells","volume":"28","author":"Yan","year":"2007","journal-title":"Acta Pharmacol. Sin."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1016\/j.toxicon.2005.01.009","article-title":"Inhibitory effect of phospholipase A2 isolated from crotalus durissus terrificus venom on macrophage function","volume":"45","author":"Sampaio","year":"2005","journal-title":"Toxicon"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1357","DOI":"10.1016\/j.toxicon.2008.03.004","article-title":"Crotoxin alters lymphocyte distribution in rats: Involvement of adhesion molecules and lipoxygenase-derived mediators","volume":"51","author":"Zambelli","year":"2008","journal-title":"Toxicon"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"7873257","DOI":"10.1155\/2018\/7873257","article-title":"Crotoxin isolated from crotalus durissus terrificus venom modulates the functional activity of dendritic cells via formyl peptide receptors","volume":"2018","author":"Freitas","year":"2018","journal-title":"J. Immunol. Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1016\/j.toxicon.2006.04.008","article-title":"Opiate and acetylcholine-independent analgesic actions of crotoxin isolated from crotalus durissus terrificus venom","volume":"48","author":"Zhang","year":"2006","journal-title":"Toxicon"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1016\/j.toxicon.2014.04.008","article-title":"Crotoxin from crotalus durissus terrificus snake venom induces the release of glutamate from cerebrocortical synaptosomes via N and P\/Q calcium channels","volume":"85","author":"Santos","year":"2014","journal-title":"Toxicon"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1003","DOI":"10.1016\/0041-0101(96)00064-5","article-title":"Re-examination of crotoxin-membrane interactions","volume":"34","author":"Krizaj","year":"1996","journal-title":"Toxicon"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Faure, G., Gowda, V.T., and Maroun, R.C. (2007). Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics. BMC Struct. Biol., 7.","DOI":"10.1186\/1472-6807-7-82"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2898","DOI":"10.1016\/j.jmb.2016.05.016","article-title":"Rattlesnake phospholipase A2 increases CFTR-chloride channel current and corrects \u2206F508CFTR dysfunction: Impact in cystic fibrosis","volume":"428","author":"Faure","year":"2016","journal-title":"J. Mol. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.toxicon.2016.02.002","article-title":"Neurotoxic phospholipase A2 from rattlesnake as a new ligand and new regulator of prokaryotic receptor GLIC (proton-gated ion channel from G. violaceus)","volume":"116","author":"Ostrowski","year":"2016","journal-title":"Toxicon"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"15646","DOI":"10.1016\/S0021-9258(17)40730-7","article-title":"A phospholipase A2 inhibitor from the plasma of the South American rattlesnake (Crotalus durissus terrificus). Protein structure, genomic structure, and mechanism of action","volume":"269","author":"Lin","year":"1994","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1111\/j.1432-1033.1995.tb20355.x","article-title":"Molecular structure and mechanism of action of the crotoxin inhibitor from crotalus durissus terrificus serum","volume":"227","author":"Perales","year":"1995","journal-title":"Eur. J. Biochem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"833","DOI":"10.1016\/S0300-9084(00)01175-5","article-title":"Natural inhibitors of toxic phospholipases A2","volume":"82","author":"Faure","year":"2000","journal-title":"Biochimie"},{"key":"ref_25","unstructured":"Gopalakrishnakone, P., Cruz, L.J., and Luo, S. (2017). Crotoxin from crotalus durissus terrificus and crotoxin-related proteins: Structure and function relationship. Toxins and Drug Discovery, Springer."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"11627","DOI":"10.1016\/S0021-9258(17)39077-4","article-title":"A comparison of the crystal structures of phospholipase A2 from bovine pancreas and Crotalus atrox venom","volume":"260","author":"Renetseder","year":"1985","journal-title":"J. Biol. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1016\/j.jmb.2011.07.027","article-title":"Crystal structure of crotoxin reveals key residues involved in the stability and toxicity of this potent heterodimeric \u03b2-neurotoxin","volume":"412","author":"Faure","year":"2011","journal-title":"J. Mol. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"883","DOI":"10.1002\/prot.21980","article-title":"Insights into the role of oligomeric state on the biological activities of crotoxin: Crystal structure of a tetrameric phospholipase A2 formed by two isoforms of crotoxin B from Crotalus durissus terrificus venom","volume":"72","author":"Magro","year":"2008","journal-title":"Proteins"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1541","DOI":"10.1126\/science.2274785","article-title":"Interfacial catalysis: The mechanism of phospholipase A2","volume":"250","author":"Scott","year":"1990","journal-title":"Science"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"774","DOI":"10.1016\/j.jmb.2007.05.022","article-title":"Inference of macromolecular assemblies from crystalline state","volume":"372","author":"Krissinel","year":"2007","journal-title":"J. Mol. Biol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"14325","DOI":"10.1021\/bi971200z","article-title":"Mapping the interfacial binding surface of human secretory group IIa phospholipase A2","volume":"36","author":"Snitko","year":"1997","journal-title":"Biochemistry"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Zambelli, V.O., Picolo, G., Fernandes, C.A.H., Fontes, M.R.M., and Cury, Y. (2017). Secreted phospholipases A2 from animal venoms in pain and analgesia. Toxins, 9.","DOI":"10.3390\/toxins9120406"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1482","DOI":"10.1016\/j.biochi.2009.08.006","article-title":"Identification of continuous interaction sites in PLA2-based protein complexes by peptide arrays","volume":"91","author":"Magro","year":"2009","journal-title":"Biochimie"},{"key":"ref_34","first-page":"671","article-title":"Structural and functional characterization of anticoagulant, FXa-binding Viperidae snake venom phospholipases A2","volume":"58","author":"Faure","year":"2011","journal-title":"Acta Chim. Slov."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Kini, R.M., Clemetson, K.J., Markland, F.S., McLane, M.A., and Morita, T. (2010). Anticoagulant phospholipases A2 which bind to the specific soluble receptor coagulation factor Xa. Toxins and Hemostasis: From Bench to Bedside, Springer.","DOI":"10.1007\/978-90-481-9295-3"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"42","DOI":"10.3109\/15569543.2013.860170","article-title":"Human coagulation factor Xa prevents oligomerization of anti-coagulant phospholipases A2","volume":"33","author":"Ostrowski","year":"2014","journal-title":"Toxin Rev."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"12535","DOI":"10.1021\/bi051024r","article-title":"Ammodytoxins, potent presynaptic neurotoxins, are also highly efficient phospholipase A2 enzymes","volume":"44","author":"Petan","year":"2005","journal-title":"Biochemistry"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1016\/j.jsb.2009.10.010","article-title":"Comparative structural studies of two natural isoforms of ammodytoxin, phospholipases A2 from Vipera ammodytes ammodytes which differ in neurotoxicity and anticoagulant activity","volume":"169","author":"Saul","year":"2010","journal-title":"J. Struct. Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"12616","DOI":"10.1016\/S0021-9258(18)33556-7","article-title":"Catalytic activity and reactivity with p-bromophenacyl bromide of the phospholipase subunit of crotoxin. Influence of dimerization and association with the noncatalytic subunit","volume":"257","author":"Radvanyi","year":"1982","journal-title":"J. Biol. Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1016\/j.toxicon.2012.05.009","article-title":"Crystallographic characterization of functional sites of crotoxin and ammodytoxin, potent \u03b2-neurotoxins from Viperidae venom","volume":"60","author":"Faure","year":"2012","journal-title":"Toxicon"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1038\/s41598-018-36461-6","article-title":"The neurotoxic secreted phospholipase A2 from the Vipera a. ammodytes venom targets cytochrome c oxidase in neuronal mitochondria","volume":"9","author":"Petan","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"4960","DOI":"10.1046\/j.1432-1327.2000.01523.x","article-title":"Basic residues of human group IIA phospholipase A2 are important for binding to factor Xa and prothrombinase inhibition comparison with other mammalian secreted phospholipases A2","volume":"267","author":"Mounier","year":"2000","journal-title":"Eur. J. Biochem."},{"key":"ref_43","first-page":"1","article-title":"Extracellular phospholipase A2 expression and inflammation: The relationship with associated disease states","volume":"8","author":"Vadas","year":"1993","journal-title":"J. Lipid Mediat."},{"key":"ref_44","unstructured":"Faure, G., and Saul, F. (2008, January 20\u201321). Caract\u00e9ristiques structurales et fonctionnelles de deux\u03b2-neurotoxines: L\u2019ammodytoxine et la crotoxine. Proceedings of the Toxines et Fonctions Cholinergiques Neuronales et non Neuronales, Paris, France."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1111\/j.1432-1033.1994.tb18978.x","article-title":"The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus","volume":"223","author":"Faure","year":"1994","journal-title":"Eur. J. Biochem."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Weber, P., Pissis, C., Navaza, R., Mechaly, A.E., Saul, F., Alzari, P.M., and Haouz, A. (2019). High-throughput crystallization pipeline at the crystallography core facility of the institut pasteur. Molecules, 24.","DOI":"10.3390\/molecules24244451"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1107\/S0907444909047374","article-title":"Integration, scaling, space-group assignment and post-refinement","volume":"66","author":"Kabsch","year":"2010","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1107\/S0907444910045749","article-title":"Overview of the CCP4 suite and current developments","volume":"67","author":"Winn","year":"2011","journal-title":"Acta Crystallogr. D Biol. Crystallogr."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"658","DOI":"10.1107\/S0021889807021206","article-title":"Phaser crystallographic software","volume":"40","author":"McCoy","year":"2007","journal-title":"J Appl. Crystallogr."},{"key":"ref_50","unstructured":"Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, P., Roversi, P., Sharff, A., Smart, O., and Vonrhein, C. (2010). BUSTER, Global Phasing Ltd."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"486","DOI":"10.1107\/S0907444910007493","article-title":"Features and development of Coot","volume":"66","author":"Emsley","year":"2010","journal-title":"Acta Crystallogr. D Biol. Crystallogr."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/25\/22\/5290\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:32:51Z","timestamp":1760178771000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/25\/22\/5290"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,11,13]]},"references-count":51,"journal-issue":{"issue":"22","published-online":{"date-parts":[[2020,11]]}},"alternative-id":["molecules25225290"],"URL":"https:\/\/doi.org\/10.3390\/molecules25225290","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,11,13]]}}}