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

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.

The interaction between the natural metalloendopeptidase inhibitor BJ46a and its target toxin jararhagin analyzed by structural mass spectrometry and molecular modeling.

Snakebite envenoming affects millions of people worldwide, being officially considered a neglected tropical disease by the World Health Organization. The antivenom is effective in neutralizing the systemic effects of envenomation, but local effects are poorly neutralized, often leading to permanent disability. The natural resistance of the South American pit viper Bothrops jararaca to its venom is partly attributed to BJ46a, a natural snake venom metalloendopeptidase inhibitor. Upon complex formation, BJ46a binds non-covalently to the metalloendopeptidase, rendering it unable to exert its proteolytic activity. However, the structural features that govern this interaction are largely unknown. In this work, we applied structural mass spectrometry techniques (cross-linking-MS and hydrogen-deuterium exchange MS) and in silico analyses (molecular modeling, docking, and dynamics simulations) to understand the interaction between BJ46a and jararhagin, a metalloendopeptidase from B. jararaca venom. We explored the distance restraints generated from XL-MS experiments to guide the modeling of BJ46a and jararhagin, as well as the protein-protein docking simulations. HDX-MS data pinpointed regions of protection/deprotection at the interface of the BJ46a-jararhagin complex which, in addition to the molecular dynamics simulation data, reinforced our proposed interaction model. Ultimately, the structural understanding of snake venom metalloendopeptidases inhibition by BJ46a could lead to the rational design of drugs to improve anti-snake venom therapeutics, alleviating the high morbidity rates currently observed.

Ferritin from the haemolymph of adult ants: an extraction method for characterization and a ferromagnetic study.

Ferritin has been studied in many animals, plants and bacteria. The main functions of ferritin in mammals are iron concentration and stabilization, protection against oxidants and iron storage for later developmental or iron-dependent activities. Although insect ferritin plays a key role in iron transport, only a few studies to date have examined its properties and function. Ferritin isolation from the haemolymph of adult Camponotus sericeiventris ants involved heating at 75 °C, followed by protein fractionation with 3.2 M KBr gradients and ferritin sedimentation with KBr. Protein identification was performed using high-resolution proteomics techniques. SDS-PAGE revealed three subunits with molecular weights (MW) of 26, 28 and 31 kDa. Native PAGE indicated a MW higher than 669 kDa. Proteomic analysis strongly suggested the 26 and 31 kDa bands as F2LCH and F1HCH subunits of ferritin, respectively. Ferromagnetic resonance (FMR) at 100 K showed, at low field, a characteristic broad component of the ferritin iron core, suggesting that its distribution was shifted to values greater than 3000, a higher content than in mammals. The protein yield and MW were comparable to those reported in other studies of insects. To the best of our knowledge, this is the first report on ferritin extracted from adult ants to date. These results are discussed on the basis of the protein structure-function relation of secreted insect and mammal ferritins. This purification method will allow the use of magnetic techniques, which are relevant for understanding the role of ferritin in the biomineralization of magnetic nanoparticles in insects.