Identification of the protein receptor binding site of botulinum neurotoxins B and G oroves the double-receptor concept
Identification of the protein receptor binding site of botulinum neurotoxins B and G oroves the double-receptor concept
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DOI：
10.1073/pnas.0609713104
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发表时间：
2007-01-02
影响因子：
11.1
通讯作者：
Binz, Thomas
中科院分区：
文献类型：
--
作者：
Rummel, Andreas;Eichner, Timo;Binz, Thomas
肉毒神经毒素（BoNT）通过选择性地裂解运动神经元内囊泡融合机制的核心成分而引起肌肉麻痹。复杂的神经节苷脂最初结合到一个口袋里，这是保守的七个BoNT和破伤风神经毒素。生产性神经毒素摄取也需要蛋白质受体。神经毒素内蛋白质受体的相互作用位点目前尚不清楚。本文报道了BoNT/B和BoNT/G的蛋白受体结合位点的鉴定和表征。它们的蛋白质受体，突触结合肽I和II，结合到H-cc（重链C-末端片段的C-末端结构域）顶端的口袋，该口袋对应于破伤风神经毒素的独特的第二碳水化合物结合位点，唾液酸结合位点。在这一地区的氨基酸取代受损的结合突触结合蛋白和小鼠膈神经制剂的毒性急剧下降; CD-光谱分析证明，突变的神经毒素的二级结构是不变的。失活的synaptotagmin结合位点的单一突变导致几乎无活性的BoNT/B和BoNT/G时，在膈神经制备的复杂神经节苷脂缺陷小鼠进行测定。类似地，BoNT B突变体与失活的神经节苷脂和突触结合蛋白结合位点缺乏明显的活性，在野生型小鼠膈神经制剂。因此，这些数据排除了除了一种神经节苷脂和一种蛋白质受体之外的任何细胞表面分子对BoNT进入过程的相关贡献，这证实了双受体概念。突触结合蛋白结合位点的分子表征为设计一类新的有效结合抑制剂提供了基础。
Botulinum neurotoxins (BoNTs) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery within motoneurons. Complex gangliosides initially bind into a pocket that is conserved among the seven BoNTs and tetanus neurotoxin. Productive neurotoxin uptake also requires protein receptors. The interaction site of the protein receptor within the neurotoxin is currently unknown. We report the identification and characterization of the protein receptor binding site of BoNT/B and BoNT/G. Their protein receptors, synaptotagimins I and II, bind to a pocket at the tip of their H-cc (C-terminal domain of the C-terminal fragment of the heavy chain) that corresponds to the unique second carbohydrate binding site of tetanus neurotoxin, the sialic acid binding site. Substitution of amino acids in this region impaired binding to synaptotagmins and drastically decreased toxicity at mouse phrenic nerve preparations; CD-spectroscopic analyses evidenced that the secondary structure of the mutated neurotoxins was unaltered. Deactivation of the synaptotagmin binding site by single mutations led to virtually inactive BoNT/B and BoNT/G when assayed at phrenic nerve preparations of complex-ganglioside-deficient mice. Analogously, a BoNT B mutant with deactivated ganglioside and synaptotagmin binding sites lacked appreciable activity at wild-type mouse phrenic nerve preparations. Thus, these data exclude relevant contributions of any cell surface molecule other than one ganglioside and one protein receptor to the entry process of BoNTs, which substantiates the double-receptor concept. The molecular characterization of the synaptotagmin binding site provides the basis for designing a novel class of potent binding inhibitors.