Structural and functional evidence for membrane docking and disruption sites on phospholipase A2-like proteins revealed by complexation with the inhibitor suramin

Local myonecrosis resulting from snakebite envenomation is not efficiently neutralized by regular antivenom administration. This limitation is considered to be a significant health problem by the World Health Organization. Phospholipase A2-like (PLA2-like) proteins are among the most important proteins related to the muscle damage resulting from several snake venoms. However, despite their conserved tertiary structure compared with PLA2s, their biological mechanism remains incompletely understood. Different oligomeric conformations and binding sites have been identified or proposed, leading to contradictory data in the literature. In the last few years, a comprehensive hypothesis has been proposed based on fatty-acid binding, allosteric changes and the presence of two different interaction sites. In the present study, a combination of techniques were used to fully understand the structural-functional characteristics of the interaction between suramin and MjTX-II (a PLA2-like toxin). In vitro neuromuscular studies were performed to characterize the biological effects of the protein-ligand interaction and demonstrated that suramin neutralizes the myotoxic activity of MjTX-II. The high-resolution structure of the complex identified the toxin-ligand interaction sites. Calorimetric assays showed two different binding events between the protein and the inhibitor. It is demonstrated for the first time that the inhibitor binds to the surface of the toxin, obstructing the sites involved in membrane docking and disruption according to the proposed myotoxic mechanism. Furthermore, higher-order oligomeric formation by interaction with interfacial suramins was observed, which may also aid the inhibitory process. These results further substantiate the current myotoxic mechanism and shed light on the search for efficient inhibitors of the local myonecrosis phenomenon.Peer Reviewe

DNA mismatch repair proteins MLH1 and PMS2 can be imported to the nucleus by a classical nuclear import pathway

MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The individual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed.Peer Reviewe

Comparative Biochemical Studies Of Myotoxic Phospholipase A2 From Bothrops Venom

Venoms from Bothrops jararacussu, Bothrops asper, Bothrops atrox, Bothrops pirajai, Bothrops moojeni, Bothrops alternatus and Bothrops (Bothriopsis) bilineata were fractionated using a simplified procedure based on ion-exchange chromatography on CM-Sepharose at pH 8.0 or reverse phase HPLC. The resulting elution profiles showed important differences in the myotoxin content of these venoms. The venoms from B. alternatus, B. atrox and Bothriopsis bilineata did not contain the major myotoxin found in the other venoms. The amino acid sequence of the first 50 residues of the N-terminal region of the PLA2-like myotoxins showed a homology of 90-96% with other bothropic myotoxins. All of the myotoxins isolated induced rat paw edema, increased the level of plasma creatine kinase and produced myonecrosis together with polymorphonuclear cell infiltration.83179186Hoge, A.R., Romano Hoge, S.A.R.W.L., (1978) Mem. Inst. Butantan, 42-43, pp. 373-496Chang, L.-S., Kuo, K.-W., Lin, S.-R., Chang, C.-C., (1994) J. Protein Chem., 13, pp. 641-648Shimohigashi, Y., Tani, A., Matsumoto, H., Nakashima, K.-I., Yamaguchi, Y., Oda, N., Takano, Y., Ohno, M., (1995) J. Biochem. (Tokyo) Biochem., 118, pp. 1037-1044Ogawa, T., Nakashima, K.-I., Nobushima, I., Deshimaru, M., Shimohigashi, Y., Fukumaki, Y., Sakaki, Y., Ohno, M., (1996) Toxicon, 34, pp. 1229-1236Dennis, E.A., (1994) Biol. Chem., 269, pp. 13057-13060Araújo, H.S., White, S.P., Ownby, C.L., (1996) Toxicon, 34, pp. 1237-1242Kini, R.M., Iwanaga, S., (1986) Toxicon, 24, pp. 895-905Arni, R.K., Ward, R.J., (1996) Toxicon, 34, pp. 827-841Mancuso, L.C., Correa, M.M., Vieira, C.A., Cunha, O.A.B., Lachat, J.J., Selistre, H.S.A., Ownby, C.L., Giglio, J.R., (1995) Toxicon, 33, pp. 615-626Soares, A.M., Rodrigues, V.M., Homsi-Brandeburgo, M.I., Toyama, M.H., Lombard, F.R., Arni, A.K., Giglio, J.R., (1998) Toxicon, 36, pp. 503-514Gutiérrez, J.M., Lomonte, B., (1995) Toxicon, 33, pp. 1405-1424Fletcher, J.E., Humbert, M., Wieland, S.J., Gong, Q.H., Jiang, M.S., (1996) Toxicon, 34, pp. 1301-1311Cho, W., Kezdy, F.J., (1991) Methods Enzymol., 197, pp. 75-79Holzer, M., Mackessy, S.P., (1996) Toxicon, 35, pp. 1149-1155Marangoni, S., Toyama, M.H., Arantes, E.C., Giglio, J.R., Da Silva, C.A., Carneiro, E.M., Gonçalves, A.A., Oliveira, B., (1995) Biophys. Acta, 1243, pp. 309-314Higgins, D.G., Sharp, P.M., (1989) Comput. Appl. Biosci., 5, pp. 151-153Araújo, A.L., Radvanyi, F., Bon, C., (1994) Toxicon, 32, pp. 1069-1081Fukagawa, T., Nose, T., Shimohigashi, Y., Ogawa, T., Oda, N., Nakashima, K.I., Chang, C.C., Ohno, M., (1993) Toxicon, 31, pp. 957-967Homsi-Brandeburgo, M.I., Queiroz, L.S., Santo-Neto, H., Rodrigues-Simioni, L., Giglio, J.R., (1988) Toxicon, 26, pp. 615-627Nakai, M., Nakashima, K.I., Ogawa, T., Shimohigashi, Y., Hattori, S., Chang, C.C., Ohno, M., (1995) Toxicon, 33, pp. 1469-1478Toyama, M.H., Soares, A.M., Vieira, C.A., Novello, J.C., Oliveira, B., Giglio, J.R., Marangoni, S., (1998) J. Protein Chem., 17, pp. 713-718Soares, A.M., Anzaloni Pedrosa, L.H., Fontes, M.R.M., Da Silva, R.J., Giglio, J.R., (1998) J. Venom. Anim. Toxins, 4. , in pressDe Azevedo W.F., Jr., Ward, R.J., Gutierrez, J.M., Arni, R.K., (1998) Toxicon, pp. 1395-1406Francis, B., Gutiérrez, J.M., Lomonte, B., Kaiser, I.I., (1991) Arch. Biochem. Biophys., 284, pp. 352-359Toyama, M.H., Mancuso, L.C., Giglio, J.R., Novello, J.C., Oliveira, B., Marangoni, S., (1995) Biochem. Mol. Biol. Inter., 37, pp. 1047-1055Heirikson, R.L., Trueger, E.T., Kein, P.S., (1977) J. Biol. Chem., 252, pp. 4913-4921Lomonte, B., Gutiérrez, J.M., Mata, E., (1985) Toxicon, 23, pp. 807-813Cintra, A.C.O., Marangoni, S., Oliveira, B., Giglio, J.R., (1993) J. Protein Chem., 12, pp. 57-64Kaiser, I.I., Gutiérrez, J.M., Plummer, D., Aird, S.D., Odell, G.V., (1990) Arch. Biochem. Biophys., 278, pp. 319-325Gowda, V.T., Schmidt, J., Middlebrook, J.L., (1994) Toxicon, 32, pp. 665-67

Functional and structural studies of a Phospholipase A2-like protein complexed to zinc ions: Insights on its myotoxicity and inhibition mechanism

One of the main challenges in snakebite envenomation treatment is the development of stable, versatile and efficient anti-venom therapies. Local myotoxicity in accidents involving snakes from the Bothrops genus is still a consequence of serum therapy inefficient neutralization that may lead to permanent sequelae in their victims. One of the classes of toxins that participate in muscle necrosis is the PLA2-like proteins. The aim of this work was to investigate the role of zinc ions in the inhibition of PLA2-like proteins and to advance the current knowledge of their action mechanism. Methods Myographic and electrophysiological techniques were used to evaluate the inhibitory effect of zinc ions, isothermal titration calorimetry assays were used to measure the affinity between zinc ions and the toxin and X-ray crystallography was used to reveal details of this interaction. Results We demonstrated that zinc ions can effectively inhibit the toxin by the interaction with two different sites, which are related to two different mechanism of inhibition: preventing membrane disruption and impairing the toxin state transition. Furthermore, structural study presented here included an additional step in the current myotoxic mechanism improving the comprehension of the allosteric transition that PLA2-like proteins undergo to exert their function. Conclusions Our findings show that zinc ions are inhibitors of PLA2-like proteins and suggest two different mechanisms of inhibition for these ions. General significance Zinc is a new candidate that can assist in anti-venom treatments and can promote the design of new and even more accurate structure-based inhibitors for PLA2-like proteins.1861131993209CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP300596/2013-81592/20112015/17286-0; 2013/00873-

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. 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