Substrate recognition strategy for botulinum neurotoxin serotype A
Substrate recognition strategy for botulinum neurotoxin serotype A
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DOI：
10.1038/nature03123
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发表时间：
2004-12-16
期刊：
影响因子：
64.8
通讯作者：
Brunger, AT
中科院分区：
文献类型：
--
作者：
Breidenbach, MA;Brunger, AT
梭菌神经毒素（CNT）是神经麻痹性疾病肉毒中毒和破伤风的病原体（1，2）。CNT通过称为SNARE的必需蛋白质的特异性蛋白水解损害神经元胞吐作用（3）。据信SNARE组装成低能三元复合物催化膜融合，促使神经递质释放;该过程响应于SNARE蛋白水解而减弱（4-7）。位点特异性SNARE水解由CNT轻链催化，CNT轻链是一组独特的锌依赖性内肽酶（3）。CNT正确识别和切割其靶SNARE的方法一直是许多推测的主题;认为其使用远离活性位点（外切位点）的一个或多个酶底物相互作用区域（8-10）。在这里，我们报告的第一个结构的CNT内肽酶在复杂的目标陷阱在2.1埃的分辨率：肉毒杆菌神经毒素血清型A（BoNT/A）蛋白酶结合到人类SNAP-25。的结构，连同酶的动力学数据，揭示了一系列的外切位点，确定底物的特异性。底物方向与一般锌依赖性金属蛋白酶嗜热菌蛋白酶的方向相似（11）。我们观察到显着的结构变化附近的毒素的催化口袋底物结合后，可能服务于使蛋白酶催化能力。这些新结构的底物识别外位点可用于设计BoNT/A特异性抑制剂。
Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus(1,2). CNTs impair neuronal exocytosis through specific proteolysis of essential proteins called SNAREs(3). SNARE assembly into a low-energy ternary complex is believed to catalyse membrane fusion, precipitating neurotransmitter release; this process is attenuated in response to SNARE proteolysis(4-7). Site-specific SNARE hydrolysis is catalysed by the CNT light chains, a unique group of zinc-dependent endopeptidases(3). The means by which a CNT properly identifies and cleaves its target SNARE has been a subject of much speculation; it is thought to use one or more regions of enzyme substrate interaction remote from the active site (exosites)(8-10). Here we report the first structure of a CNT endopeptidase in complex with its target SNARE at a resolution of 2.1 Angstrom: botulinum neurotoxin serotype A (BoNT/A) protease bound to human SNAP-25. The structure, together with enzyme kinetic data, reveals an array of exosites that determine substrate specificity. Substrate orientation is similar to that of the general zinc-dependent metalloprotease thermolysin(11). We observe significant structural changes near the toxin's catalytic pocket upon substrate binding, probably serving to render the protease competent for catalysis. The novel structures of the substrate-recognition exosites could be used for designing inhibitors specific to BoNT/A.