Molecular Architecture of the Antiophidic Protein DM64 and its Binding Specificity to Myotoxin II From Bothrops asper Venom.
Molecular Architecture of the Antiophidic Protein DM64 and its Binding Specificity to Myotoxin II From Bothrops asper Venom.
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抗嗜蛋蛋白DM64的分子结构及其与Bothrops Asper毒液中肌毒素II的结合特异性。
DOI：
10.3389/fmolb.2021.787368
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发表时间：
2021
影响因子：
5
通讯作者：
Neves-Ferreira AGC
中科院分区：
文献类型：
--
作者：
Soares BS;Rocha SLG;Bastos VA;Lima DB;Carvalho PC;Gozzo FC;Demeler B;Williams TL;Arnold J;Henrickson A;Jørgensen TJD;Souza TACB;Perales J;Valente RH;Lomonte B;Gomes-Neto F;Neves-Ferreira AGC
DM 64是从Didelphis aurita分离的毒素中和血清糖蛋白，Didelphis aurita是一种天然抗蛇毒的食蛇有袋类动物。这种64 kDa的抗毒素靶向肌毒性磷脂酶A2，其导致蝰蛇咬伤的大多数局部组织损伤。我们研究了天然DM 64和肌毒素II之间形成的非共价复合物，肌毒素II是一种来自Bothrops asper毒液的肌毒性磷脂酶样蛋白。分析超速离心（AUC）和尺寸排阻色谱表明DM 64在溶液中是单体，并结合等摩尔量的毒素。尝试结晶天然DM 64用于X射线衍射是不成功的。获得重组蛋白以进行结构研究也具有挑战性。由于缺乏与DM 64具有超过25%序列同一性的模板，经典的分子建模技术受到损害。一个综合的结构生物学方法，然后应用到生成一个三维模型的抑制剂结合到myotoxin II。I-TASSER分别模拟了DM 64的五个免疫球蛋白样结构域。距离限制产生的复合物的交联质谱指导对接的DM 64域的肌毒素II的晶体结构，使用Rosetta。AUC、小角X射线散射（SAXS）、分子建模和分子动力学模拟表明，DM 64-肌毒素II复合物是结构化的，显示出柔性，并且具有各向异性形状。蛋白质间的交联和有限的水解分析揭示了与毒素相互作用的抑制剂的区域，揭示了DM 64的第一，第三和第五结构域的关键参与。我们的数据显示，DM 64的第五结构域结合肌毒素II氨基末端和β翼区。抑制剂的第三结构域以与第五结构域互补的方式起作用。它们与这些毒素区域的结合可能排除了二聚化，从而干扰了毒性，这与毒素的四级结构有关。DM 64的第一个结构域与与膜锚定相关的毒素puppet的功能位点相互作用。我们提出，这两种机制一致抑制肌毒素II的毒性DM 64结合。目前的拓扑特性，这种毒素-抗毒素复合物构成了一个重要的一步，以合理的设计新的肽为基础的抗蛇毒疗法，针对蛇毒肌毒素。
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.
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影响因子：
3.4
作者：
Cao, Weiming;Demeler, Borries
通讯作者：
Demeler, Borries
影响因子：
4.2
作者：
Camacho E;Sanz L;Escalante T;Pérez A;Villalta F;Lomonte B;Neves-Ferreira AG;Feoli A;Calvete JJ;Gutiérrez JM;Rucavado A
通讯作者：
Rucavado A
影响因子：
4.2
作者：
Bastos VA;Gomes-Neto F;Perales J;Neves-Ferreira AG;Valente RH
通讯作者：
Valente RH
DOI：
10.1107/s0907444994011455
发表时间：
1995-05-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
ARNI, RK;WARD, RJ;TULINSKY, A
通讯作者：
TULINSKY, A
影响因子：
1.6
作者：
dos Santos, Juliana I.;Fernandes, Carlos A. H.;Fontes, Marcos R. M.
通讯作者：
Fontes, Marcos R. M.