Structure- and substrate-based inhibitor design for Clostridium botulinum neurotoxin serotype A
Structure- and substrate-based inhibitor design for Clostridium botulinum neurotoxin serotype A
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DOI：
10.1074/jbc.m801240200
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发表时间：
2008-07-04
影响因子：
4.8
通讯作者：
Swaminathan, Subramanyam
中科院分区：
文献类型：
--
作者：
Kumaran, Desigan;Rawat, Richa;Swaminathan, Subramanyam
肉毒梭菌神经毒素的七种抗原性不同血清型切割特异性可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体复合物蛋白，并阻断引起弛缓性麻痹的神经递质的释放，被认为是潜在的生物武器。A型肉毒杆菌神经毒素是梭菌神经毒素中效力最强的一种，迄今为止还没有可用的接触后治疗干预措施。为了开发导致药物设计的抑制剂，必须知道酶与活性位点附近的底物之间的关键相互作用。虽然酶-底物相互作用的exosites远离活性位点的详细映射A型肉毒杆菌神经毒素，活性位点的相互作用的信息是缺乏的。本文报道了A型肉毒神经毒素催化结构域与四种抑制底物类似物四肽（RRGC、RRGL、RRGI和RRGM）复合物的晶体结构，分辨率为1.6-1.8 A。这些结构首次显示了底物和酶在活性位点的相互作用，并描述了对底物稳定和催化活性重要的残基。我们发现Tyr（366）的OH和Arg（363）的NH 2与底物类似物的P1和P1'的羰基氧形成氢键，并定位其催化活性。最重要的是，亲核水被四肽N末端残基的氨基取代。此外，S1'位点由Phe（194）、Thr（215）、Thr（220）、Asp（370）和Arg（363）形成。最佳抑制四肽的Ki为157 nM。
The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins cleave specific soluble N-ethylmaleimidesensitive factor attachment protein receptor complex proteins and block the release of neurotransmitters that cause flaccid paralysis and are considered potential bioweapons. Botulinum neurotoxin type A is the most potent among the clostridial neurotoxins, and to date there is no post-exposure therapeutic intervention available. To develop inhibitors leading to drug design, it is imperative that critical interactions between the enzyme and the substrate near the active site are known. Although enzyme-substrate interactions at exosites away from the active site are mapped in detail for botulinum neurotoxin type A, information about the active site interactions is lacking. Here, we present the crystal structures of botulinum neurotoxin type A catalytic domain in complex with four inhibitory substrate analog tetrapeptides, viz. RRGC, RRGL, RRGI, andRRGM at resolutions of 1.6-1.8 A. These structures show for the first time the interactions between the substrate and enzyme at the active site and delineate residues important for substrate stabilization and catalytic activity. We show that OH of Tyr(366) and NH2 of Arg(363) are hydrogen-bonded to carbonyl oxygens of P1 and P1' of the substrate analog and position it for catalytic activity. Most importantly, the nucleophilic water is replaced by the amino group of the N-terminal residue of the tetrapeptide. Furthermore, the S1' site is formed by Phe(194), Thr(215), Thr(220), Asp(370), and Arg(363). The K-i of the best inhibitory tetrapeptide is 157 nM.