Structural Bases For A Complete Myotoxic Mechanism: Crystal Structures Of Two Non-catalytic Phospholipases A2-like From Bothrops Brazili Venom

Bothrops brazili is a snake found in the forests of the Amazonian region whose commercial therapeutic anti-bothropic serum has low efficacy for local myotoxic effects, resulting in an important public health problem in this area. Catalytically inactive phospholipases A2-like (Lys49-PLA 2s) are among the main components from Bothrops genus venoms and are capable of causing drastic myonecrosis. Several studies have shown that the C-terminal region of these toxins, which includes a variable combination of positively charged and hydrophobic residues, is responsible for their activity. In this work we describe the crystal structures of two Lys49-PLA2s (BbTX-II and MTX-II) from B. brazili venom and a comprehensive structural comparison with several Lys49-PLA2s. Based on these results, two independent sites of interaction were identified between protein and membrane which leads to the proposition of a new myotoxic mechanism for bothropic Lys49-PLA2s composed of five different steps. This proposition is able to fully explain the action of these toxins and may be useful to develop efficient inhibitors to complement the conventional antivenom administration. © 2013 Elsevier B.V. All rights reserved.18341227722781Campbell, J.A., Lamar, W.W., (2004) The Venomous Reptiles of the Western Hemisphere, , Comstock Press New YorkMuniz, E.G., Maria, W.S., Estevao-Costa, M.I., Buhrnheim, P., Chavez-Olortegui, C., Neutralizing potency of horse antibothropic Brazilian antivenom against Bothrops snake venoms from the Amazonian rain forest (2000) Toxicon, 38 (12), pp. 1859-1863. , DOI 10.1016/S0041-0101(00)00082-9, PII S0041010100000829Kasturiratne, A., Wickresmasinghe, A.R., De Silva, N., Gunawaderna, N.K., Pathmeswaran, A., Premaratna, R., Savioli, L., De Silva, J., The global burden of snakebite: A literature analysis and modelling based on regional estimates of envenoming and deaths (2008) Plos Med., 5, p. 218Williams, D., Gutiérrez, J.M., Harrison, R., Warrell, D.A., White, J., Winkel, K.D., Gopalakrishnakone, P., The Global Snake Bite Initiative: An antidote for snakebite (2010) Lancet, 375, pp. 89-91Araújo, F.A.A., Santalúcia, M., Cabra, R.F., Epidemiologia dos acidentes por animais peçonhentos (2003) Animais Peçonhentos No Brasil, pp. 06-12. , J.L.C. Cardoso, F.O.S. França, F.H. Wen, C.M.S. Málaque, V. Haddad Jr. Sarvier São PauloSaúde, F.N., (2001) Manual de Diagnóstico e Tratamento de Acidentes Por Animais Peçonhentos, , FUNASA BrasíliaGutierrez, J.M., Lomonte, B., Phospholipase A2 myotoxins from Bothrops snake venoms (1995) Toxicon, 33 (11), pp. 1405-1424. , DOI 10.1016/0041-0101(95)00085-ZGutiérrez, J.M., Theakston, R.D.G., Warrell, D.A., Confronting the neglected problem of snake bite envenoming: The need for a global partnership (2006) Plos Med., 3, p. 150Verheij, H.M., Volwerck, J.J., Jansen, E.H., Puyk, W.C., Dijikstra, B.W., Drenth, J., De Haas, G.H., Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism (1980) Biochemistry, 19, pp. 743-750Scott, D.L., White, S.P., Otwinowski, Z., Yuan, W., Gelb, M.H., Sigler, P.B., Interfacial catalysis: The mechanism of phospholipase A2 (1990) Science, 250 (4987), pp. 1541-1546Holland, D.R., Clancy, L.L., Muchmore, S.W., Ryde, T.J., Eispahr, H.M., Finzel, B.C., Heinrikson, R.L., Watenpaugh, K.D., The crystal structure of a lysine 49 phospholipase A2 from the venom of the cottonmouth snake at 2.0 Å resolution (1990) J. Biol. Chem., 265, pp. 17649-17656Fernandes, C.A., Marchi-Salvador, D.P., Salvador, G.M., Silva, M.C., Costa, T.R., Soares, A.M., Fontes, M.R.M., Comparison between apo and complexed structures of bothropstoxin-I reveals the role of Lys122 and Ca2 + binding loop for the catalytically inactive Lys49-PLA2s (2010) J. Struct. Biol., 171, pp. 31-43Arni, R.K., Ward, R.J., Phospholipase A2 - A structural review (1996) Toxicon, 34 (8), pp. 827-841. , DOI 10.1016/0041-0101(96)00036-0Lomonte, B., Moreno, E., Tarkowski, A., Hanson, L.A., Maccarana, M., Neutralizing interaction between heparins and myotoxin II, a lysine 49 phospholipase A2 from Bothrops asper snake venom. Identification of a heparin-binding and cytolytic toxin region by the use of synthetic peptides and molecular modeling (1994) J. Biol. Chem., 269, pp. 29867-29873Falconi, M., Desideri, A., Rufini, S., Membrane-perturbing activity of Viperidae myotoxins: An electrostatic surface potential approach to a puzzling problem (2000) Journal of Molecular Recognition, 13 (1), pp. 14-19. , DOI 10.1002/(SICI)1099-1352(200001/02)13: 13.0. CO;2-FNuez, C.E., Angulo, Y., Lomonte, B., Identification of the myotoxic site of the Lys49 phospholipase A 2 from Agkistrodon piscivorus piscivorus snake venom: Synthetic C-terminal peptides from Lys49, but not from Asp49 myotoxins, exert membrane-damaging activities (2001) Toxicon, 39 (10), pp. 1587-1594. , DOI 10.1016/S0041-0101(01)00141-6, PII S0041010101001416Chioato, L., Aragao, E.A., Lopes Ferreira, T., Ivo De Medeiros, A., Faccioli, L.H., Ward, R.J., Mapping of the structural determinants of artificial and biological membrane damaging activities of a Lys49 phospholipase A2 by scanning alanine mutagenesis (2007) Biochimica et Biophysica Acta - Biomembranes, 1768 (5), pp. 1247-1257. , DOI 10.1016/j.bbamem.2007.01.023, PII S0005273607000338Da Silva Giotto, M.T., Garratt, R.C., Oliva, G., Mascarenhas, Y.P., Giglio, J.R., Cintra, A.C.O., De Azevedo Jr., W.F., Ward, R.J., Crystallographic and spectroscopic characterization of a molecular hinge: Conformational changes in Bothropstoxin I, a dimeric Lys49- phospholipase A2 homologue (1998) Proteins: Structure, Function and Genetics, 30 (4), pp. 442-454. , DOI 10.1002/(SICI)1097-0134(19980301)30: 43.0. CO;2-IMurakami, M.T., Vicoti, M.M., Abrego, J.R.B., Lourenzoni, M.R., Cintra, A.C.O., Arruda, E.Z., Tomaz, M.A., Arni, R.K., Interfacial surface charge and free accessibility to the PLA 2-active site-like region are essential requirements for the activity of Lys49 PLA2 homologues (2007) Toxicon, 49 (3), pp. 378-387. , DOI 10.1016/j.toxicon.2006.10.011, PII S0041010106004016Dos Santos, J.I., Fernandes, C.A.H., Magro, A.J., Fontes, M.R.M., The intriguing phospholipases A2 homologues: Relevant structural features on myotoxicity and catalytic inactivity (2009) Protein Pept. Lett., 16, pp. 887-893De Oliveira, A.H.C., Giglio, J.R., Andriao-Escarso, S.H., Ito, A.S., Ward, R.J., A pH-induced dissociation of the dimeric form of a lysine 49-phospholipase A2 abolishes Ca2+-independent membrane damaging activity (2001) Biochemistry, 40 (23), pp. 6912-6920. , DOI 10.1021/bi0026728Angulo, Y., Gutierrez, J.M., Soares, A.M., Cho, W., Lomonte, B., Myotoxic and cytolytic activities of dimeric Lys49 phospholipase A 2 homologues are reduced, but not abolished, by a pH-induced dissociation (2005) Toxicon, 46 (3), pp. 291-296. , DOI 10.1016/j.toxicon.2005.03.025, PII S0041010105001479Murakami, M.T., Arruda, E.Z., Melo, P.A., Martinez, A.B., Calil-Elias, S., Tomaz, M.A., Lomonte, B., Arni, R.K., Inhibition of myotoxic activity of Bothrops asper myotoxin II by the anti-trypanosomal drug suramin (2005) Journal of Molecular Biology, 350 (3), pp. 416-426. , DOI 10.1016/j.jmb.2005.04.072, PII S002228360500505XDos Santos, J.I., Soares, A.M., Fontes, M.R.M., Comparative structural studies on Lys49-phospholipases A2 from Bothrops genus reveal their myotoxic site (2009) J. Struct. Biol., 167, pp. 106-116Huancahuire-Vega, S., Ponce-Soto, L.A., Martins De Souza, D., Marangoni, S., Structural and functional characterization of brazilitoxins II and III (BbTX-II and -III), two myotoxins from the venom of Bothrops brazili snake (2009) Toxicon, 54, pp. 816-827Costa, T.R., Menaldo, D.L., Oliveira, C.Z., Santos-Filho, N.A., Teixeira, S.S., Nomizo, A., Fuly, A.L., Soares, A.M., Myotoxic phospholipases A(2) isolated from Bothops brazili snake venom and synthetic peptides derived from their C-terminal region: Cytotoxic effect on microorganism and tumor cells (2008) Peptides, 29, pp. 1645-1656Jankarik, J., Kim, S.H., Sparse-matrix sampling - A screening method for crystallization of proteins (1991) J. Appl. Crystallogr., 24, pp. 409-411Fernandes, C.A.H., Gartuzo, E.C.G., Pagotto, I., Huancahuire-Vega, S., Ponce-Soto, L.A., Costa, T.R., Marangoni, S., Fontes, M.R.M., Crystallization and preliminary X-ray diffraction analysis of three myotoxic phospholipases A2 from Bothrops brazili venom (2012) Acta Crystallogr. F, 68, pp. 935-938Otwinowski, Z., Minor, W., Processing of X-ray diffraction data collected in oscillation mode (1997) Methods in Enzymology, 276, pp. 307-326. , DOI 10.1016/S0076-6879(97)76066-XMcCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C., Read, R.J., Phaser crystallographic software (2007) Journal of Applied Crystallography, 40 (4), pp. 658-674. , DOI 10.1107/S0021889807021206, PII S0021889807021206Adams, P.D., Afonine, P.V., Bunkóczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Zwart, P.H., PHENIX: A comprehensive python-based system for macromolecular structure solution (2010) Acta Crystallogr. D, 66, pp. 213-221Emsley, P., Cowtan, K., Coot: Model-building tools for molecular graphics (2004) Acta Crystallographica Section D: Biological Crystallography, 60 (12), pp. 2126-2132. , DOI 10.1107/S0907444904019158Brunger, A.T., Adams, P.D., Clore, G.M., Delano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Warren, G.L., Crystallography and NMR system: A new software suite for macromolecular structure determination (1998) Acta Cryst. Sect. D, 54, pp. 905-921Chen, V.B., Arendall III, W.B., Headd, J.J., Keedy, D.A., Immormimo, R.M., Kapral, G.J., Murray, L.W., Richardson, D.C., MolProbity: All-atom structure validation for macromolecular crystallography (2010) Acta Crystallogr. D, 66, pp. 12-21Delano, W.S., (2002) The PyMOL Molecular Graphics System, , Delano Scientific San CarlosKrissinel, E., Henrick, K., Inference of macromolecular assemblies from crystalline state (2007) Journal of Molecular Biology, 372 (3), pp. 774-797. , DOI 10.1016/j.jmb.2007.05.022, PII S0022283607006420Watanabe, L., Soares, A.M., Ward, R.J., Fontes, M.R.M., Arni, R.K., Structural insights for fatty acid binding in a Lys49-phospholipase A 2: Crystal structure of myotoxin II from Bothrops moojeni complexed with stearic acid (2005) Biochimie, 87 (2), pp. 161-167. , DOI 10.1016/j.biochi.2004.11.005Delatorre, P., Rocha, B.A., Santi-Gadelha, T., Gadelha, C.A., Toyama, M.H., Cavada, B.S., Crystal structure of Bn IV in complex with myristic acid: A Lys49 myotoxin phospholipase A2 from Bothrops neuwiedi venom (2011) Biochimie, 93, pp. 513-518Salvador, G.H.M., Cavalcante, W.L., Dos Santos, J.I., Gallacci, M., Soares, A.M., Fontes, M.R.M., Structural and functional studies with myotoxin II from Bothrops moojeni reveal remarkable similarities and differences compared to other catalytically inactive phospholipases A2-like (2013) Toxicon, 72, pp. 52-63Pan, Y.H., Epstein, T.M., Jain, M.K., Bahnson, B.J., Five coplanar anion binding sites on one face of phospholipase A 2: Relationship to interface binding (2001) Biochemistry, 40 (3), pp. 609-617. , DOI 10.1021/bi002514gBahnson, B.J., Structure, function and interfacial allosterism in phospholipase A2: Insight from the anion-assisted dimer (2005) Archives of Biochemistry and Biophysics, 433 (1), pp. 96-106. , DOI 10.1016/j.abb.2004.08.013, PII S0003986104004576White, S.P., Scott, D.L., Otwinowski, Z., Gelb, M.H., Sigler, P.B., Crystal structure of cobra-venom phospholipase A2 in a complex with a transition-state analogue (1990) Science, 250 (4987), pp. 1560-1563Ambrosio, A.L.B., Nonato, M.C., Selistre De Araujo, H.S., Arni, R., Ward, R.J., Ownby, C.L., De Souza, D.H.F., Garratt, R.C., A molecular mechanism for Lys49-phospholipase A2 activity based on ligand-induced conformational change (2005) Journal of Biological Chemistry, 280 (8), pp. 7326-7335. , DOI 10.1074/jbc.M410588200Murakami, M.T., Kuch, U., Betzel, C., Mebs, D., Arni, R.K., Crystal structure of a novel myotoxic Arg49-phospholipase A2 homologue (zhaoermiatoxin) from Zhaoermia mangshanensis snake venom: Insights into Arg49 coordination and the role of Lys122 in polarization of the C-terminus (2008) Toxicon, 51, pp. 723-735Chioato, L., De Oliveira, A.H., Ruller, R., Sá, J.M., Ward, R.J., Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2 (2002) Biochem. J., 366, pp. 971-976Kyte, J., Doolittle, R.F., A simple method for displaying the hydropathic character of a protein (1982) J. Mol. Biol., 157, pp. 105-132Wimley, W.C., White, S.H., Experimentally determined hydrophobicity scale for proteins at membrane interfaces (1996) Nature Structural Biology, 3 (10), pp. 842-848. , DOI 10.1038/nsb1096-842Naoi, M., Naoi, M., Shimizu, T., Permeability of amino acids into liposomes (1977) Biochimica et Biophysica Acta, 471 (2), pp. 305-310Chakrabarti, A.C., Deamer, D.W., Permeability of lipid bilayers to amino acids and phosphate (1992) Biochim. Biophys. Acta, 1111, pp. 171-177Gutierrez, J.M., Ownby, C.L., Skeletal muscle degeneration induced by venom phospholipases A 2: Insights into the mechanisms of local and systemic myotoxicity (2003) Toxicon, 42 (8), pp. 915-931. , DOI 10.1016/j.toxicon.2003.11.005Zhou, X., Tan, T.C., Valiyaveettil, S., Go, M.L., Kini, R.M., Velazquez-Campoy, A., Sivaraman, J., Structural characterization of myotoxic ecarpholin S from Echis carinatus venom (2008) Biophys. J., 95, pp. 3366-3380Yamazaki, Y., Matsunaga, Y., Nakano, Y., Morita, T., Identification of a vascular endothelial grown factor receptor-binding protein in the venom of Eastern cottonmouth (2005) J. Biol. Chem., 280, pp. 29989-29992Fujisawa, D., Yamazaki, Y., Lomonte, B., Morita, T., Catalytically inactive phospholipase A2 homologue binds to vascular endothelial growth factor receptor-2 via a C-terminal loop region (2008) Biochemical Journal, 411 (3), pp. 515-522. , DOI 10.1042/BJ20080078Cintra-Francischinelli, M., Pizzo, P., Rodrigues-Simioni, L., Ponce-Soto, L.A., Rossetto, O., Lomonte, B., Gutiérrez, J.M., Montecucco, C., Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors (2009) Cell. Mol. Life Sci., 66, pp. 1718-1728Lomonte, B., Angulo, Y., Calderon, L., An overview of lysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action (2003) Toxicon, 42 (8), pp. 885-901. , DOI 10.1016/j.toxicon.2003.11.008Bortoleto-Bugs, R.K., Bugs, M.R., Neto, A.A., Ward, R.J., A micelle nucleation model for the interaction of dodecyl sulphate with Lys49-phospholipases A2 (2007) Biophysical Chemistry, 125 (1), pp. 213-220. , DOI 10.1016/j.bpc.2006.08.002, PII S0301462206002560De Araujo, H.S.S., White, S.P., Ownby, C.L., CDNA cloning and sequence analysis of a lysine-49 phospholipase A 2 myotoxin from Agkistrodon contortrix laticinctus snake venom (1996) Archives of Biochemistry and Biophysics, 326 (1), pp. 21-30. , DOI 10.1006/abbi.1996.0042Dos Santos, J.I., Cintra-Francischinelli, M., Borges, R.J., Fernandes, C.A., Pizzo, P., Cintra, A.C., Braz, A.S., Fontes, M.R., Structural, functional and bioinformatics studies reveal a new snake venom homologue phospholipase A2 class (2011) Proteins, 79, pp. 61-78Cintra Francischinelli, M., Caccin, P., Chiavegato, A., Pizzo, P., Carmignoto, G., Ângulo, Y., Lomonte, B., Montecucco, C., Bothrops snake myotoxins induce a large efflux of ATP and potassium with spreading of cell damage and pain (2010) Proc. Natl. Acad. Sci. U. S. A., 107, pp. 14140-14145Magro, A.J., Soares, A.M., Giglio, J.R., Fontes, M.R.M., Crystal structures of BnSP-7 and BnSP-6, two Lys49-phospholipases A 2: Quaternary structure and inhibition mechanism insights (2003) Biochemical and Biophysical Research Communications, 311 (3), pp. 713-720. , DOI 10.1016/j.bbrc.2003.10.047Marchi-Salvador, D.P., Fernandes, C.A., Silveira, L.B., Soares, A.M., Fontes, M.R., Crystal structure of a phospholipase A(2) homolog complexed with p-bromophenacyl bromide reveals important structural changes associated with the inhibition of myotoxic activity (2009) Biochim. Biophys. Acta, 1794, pp. 1583-1590Fenwick, A.M., Gutberlet, R.K., Evans, J.A., Parkinson, C.K., Morphological and molecular evidence for phylogenetic classification of South American pitvipers, genera Bothops, Bothriopsis and Bothrocophias (Serpentes: Viperidade) (2009) Zool. J. Linnean. Soc., 156, pp. 617-640Renetseder, R., Brunie, S., Dijkstra, B.W., A comparison of the crystal structure of phospholipase A2 from bovine pancreas and Crotalus atrox venom (1985) Journal of Biological Chemistry, 260 (21), pp. 11627-11634Schwartz, A.S., Pachter, L., Multiple alignment by sequence annealing (2007) Bioinformatics, 23, pp. 24-e2