Molecular architecture of the antiophidic protein DM64 and its binding specificity to myotoxin II from Bothrops aasper venom

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dc.contributor.authorSoares, Barbara S.
dc.contributor.authorRocha, Surza Lucia G.
dc.contributor.authorBastos, Viviane A.
dc.contributor.authorLima, Diogo B.
dc.contributor.authorCarvalho, Paulo C.
dc.contributor.authorGozzo, Fabio C.
dc.contributor.authorDemeler, Borries
dc.contributor.authorWilliams, Tayler L.
dc.contributor.authorArnold, Janelle
dc.contributor.authorHenrickson, Amy
dc.contributor.authorJorgensen, Thomas J. D.
dc.contributor.authorSouza, Tatiana A. C. B.
dc.contributor.authorPerales, Jonas
dc.contributor.authorValente, Richard H.
dc.contributor.authorLomonte, Bruno
dc.contributor.authorGomes-Neto, Francisco
dc.contributor.authorNeves-Ferreira, Ana Gisele C.
dc.date.accessioned2022-09-09T21:52:43Z
dc.date.available2022-09-09T21:52:43Z
dc.date.issued2022
dc.descriptionOpen access article. Creative Commons Attribution 4.0 International License (CC BY 4.0) appliesen_US
dc.description.abstractDM64 is a toxin-neutralizing serum glycoprotein isolated from Didelphis aurita, an ophiophagous marsupial naturally resistant to snake envenomation. This 64 kDa antitoxin targets myotoxic phospholipases A2, which account for most local tissue damage of viperid snakebites. We investigated the noncovalent complex formed between native DM64 and myotoxin II, a myotoxic phospholipase-like protein from Bothrops asper venom. Analytical ultracentrifugation (AUC) and size exclusion chromatography indicated that DM64 is monomeric in solution and binds equimolar amounts of the toxin. Attempts to crystallize native DM64 for X-ray diffraction were unsuccessful. Obtaining recombinant protein to pursue structural studies was also challenging. Classical molecular modeling techniques were impaired by the lack of templates with more than 25% sequence identity with DM64. An integrative structural biology approach was then applied to generate a three-dimensional model of the inhibitor bound to myotoxin II. I-TASSER individually modeled the five immunoglobulin-like domains of DM64. Distance constraints generated by cross-linking mass spectrometry of the complex guided the docking of DM64 domains to the crystal structure of myotoxin II, using Rosetta. AUC, small-angle X-ray scattering (SAXS), molecular modeling, and molecular dynamics simulations indicated that the DM64-myotoxin II complex is structured, shows flexibility, and has an anisotropic shape. Inter-protein cross-links and limited hydrolysis analyses shed light on the inhibitor’s regions involved with toxin interaction, revealing the critical participation of the first, third, and fifth domains of DM64. Our data showed that the fifth domain of DM64 binds to myotoxin II amino-terminal and beta-wing regions. The third domain of the inhibitor acts in a complementary way to the fifth domain. Their binding to these toxin regions presumably precludes dimerization, thus interfering with toxicity, which is related to the quaternary structure of the toxin. The first domain of DM64 interacts with the functional site of the toxin putatively associated with membrane anchorage. We propose that both mechanisms concur to inhibit myotoxin II toxicity by DM64 binding. The present topological characterization of this toxin-antitoxin complex constitutes an essential step toward the rational design of novel peptide-based antivenom therapies targeting snake venom myotoxins.en_US
dc.description.peer-reviewYesen_US
dc.identifier.citationSoares, B. S., Rocha, S. L. G., Bastos, V. A., Lima, D. B., Carvalho, P. C., Gozzo, F. C., Demeler, B., Williams, T. L., Arnold, J., Henrickson, A., Jorgensen, T. J. D., Souza, T. A. C. B., Perales, J., Valente, R. H., Lomonte, B., Gomes-Neto, F., & Neves-Ferreira, A. G. C. (2022). Molecular architecture of the antiophidic protein DM64 and its binding specificity to myotoxin II from Bothrops asper venom. Frontiers in Molecular Biosciences, 8, Article 787368. https://doi.org/10.3389/fmolb.2021.787368en_US
dc.identifier.urihttps://hdl.handle.net/10133/6330
dc.language.isoen_USen_US
dc.publisherFrontiers Research Foundationen_US
dc.publisher.departmentDepartment of Chemistry and Biochemistryen_US
dc.publisher.facultyArts and Scienceen_US
dc.publisher.institutionOswaldo Cruz Instituteen_US
dc.publisher.institutionLeibniz Forschungsinstitut für Molekulare Pharmakologie (FMP)en_US
dc.publisher.institutionCarlos Chagas Instituteen_US
dc.publisher.institutionUniversity of Campinasen_US
dc.publisher.institutionUniversity of Texas Health Science Center at San Antonioen_US
dc.publisher.institutionUniversity of Lethbridgeen_US
dc.publisher.institutionUniversity of Montanaen_US
dc.publisher.institutionPrinceton Universityen_US
dc.publisher.institutionUniversity of Southern Denmarken_US
dc.publisher.institutionUniversity of Costa Ricaen_US
dc.publisher.urlhttps://doi.org/10.3389/fmolb.2021.787368en_US
dc.subjectCross-linking (XL)en_US
dc.subjectImmunogolubin folden_US
dc.subjectStructural biologyen_US
dc.subjectToxin neutralisationen_US
dc.subjectProtein inhibitoren_US
dc.subjectSnake envenomationen_US
dc.subjectAntiophidic activityen_US
dc.subjectDM64
dc.subject.lcshProteins--Crosslinking
dc.subject.lcshMass spectrometry
dc.subject.lcshBothrops
dc.subject.lcshPoisonous snakes--Venom
dc.subject.lcshSnakebites
dc.titleMolecular architecture of the antiophidic protein DM64 and its binding specificity to myotoxin II from Bothrops aasper venomen_US
dc.typeArticleen_US
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