Molecular Architecture of the Antiophidic Protein DM64 and its Binding Specificity to Myotoxin II From Bothrops asper Venom

Frontiers in Molecular Biosciences

Bruno Lomonte¹  Fabio C. Gozzo²  Thomas J. D. Jørgensen³  Tayler L. Williams⁴  Diogo B. Lima⁵  Amy Henrickson⁶  Borries Demeler⁷  Janelle Arnold⁸  Tatiana A. C. B. Souza⁹  Paulo C. Carvalho⁹  Richard H. Valente^1,10  Ana Gisele C. Neves-Ferreira^1,10  Viviane A. Bastos^1,10  Surza Lucia G. Rocha^1,10  Jonas Perales^1,10  Francisco Gomes-Neto^1,10  Barbara S. Soares^1,10 

[1] 0Clodomiro Picado Institute, University of Costa Rica, San José, Costa Rica;Dalton Mass Spectrometry Laboratory, University of Campinas, Campinas, Brazil;Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark;Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States;Department of Chemical Biology, Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany;Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB, Canada;Department of Chemistry and Biochemistry, University of Montana, Missoula, MT, United States;Department of Environmental Science, Princeton University, Princeton, NJ, United States;Laboratory for Structural and Computational Proteomics, Carlos Chagas Institute, Curitiba, Brazil;Laboratory of Toxinology, Oswaldo Cruz Institute, Rio de Janeiro, Brazil;

关键词: cross-linking (XL);    mass spectrometry;    immunoglobulin fold;    structural biology;    toxin neutralisation;    protein inhibitor;   

DOI  :  10.3389/fmolb.2021.787368

来源: DOAJ

【 摘 要 】

DM64 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.

【 授权许可】

Unknown