Scanning mutagenesis of a Janus-faced atracotoxin reveals a bipartite surface patch that is essential for neurotoxic function
Scanning mutagenesis of a Janus-faced atracotoxin reveals a bipartite surface patch that is essential for neurotoxic function
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DOI：
10.1074/jbc.m202297200
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发表时间：
2002-06-21
影响因子：
4.8
通讯作者：
King, GF
中科院分区：
文献类型：
--
作者：
Maggio, F;King, GF
Janus-faced神经毒素（J-ACTX）是从澳大利亚漏斗网蜘蛛毒液中分离出的一类昆虫特异性兴奋性神经毒素。除了它们的名字来源于带电残基的显著不对称分布之外，这些毒素还含有极其罕见的邻位二硫键。为了阐明这些毒素的作用机制，并提高其作为杀虫剂开发的先导化合物的实用性，我们开发了原型家族成员J-ACTX-Hv 1c的重组表达系统，并使用定点突变绘制了关键功能残基。使用一组24个突变体的丙氨酸扫描提供了蜘蛛毒素的生物活性表面的第一个完整的地图，并揭示了整个J-ACTX-Hv 1c药效团被限制为7个残基，这些残基在毒素的一个面上形成二分表面补丁。然而，主要药效团或热点仅由五个残基（Arg（8）、Pro（9）、Tyr（31）和Cys（13）-Cys（14）邻位二硫化物）形成。J-ACTX-Hv 1c中的Arg（8）-Tyr（31）二联体与Lys-（Tyr/Phe）二联体紧密重叠，而Lys-（Tyr/Phe）二联体在一系列结构不同的K+通道阻断剂中是空间保守的，这使我们推测J-ACTX可能靶向无脊椎动物的K+通道。
The Janus-faced atracotoxins (J-ACTXs) are a family of insect-specific excitatory neurotoxins isolated from the venom of Australian funnel web spiders. In addition to a strikingly asymmetric distribution of charged residues, from which their name is derived, these toxins contain an extremely rare vicinal disulfide bond. To shed light on the mechanism of action of these toxins and to enhance their utility as lead compounds for insecticide development, we developed a recombinant expression system for the prototypic family member, J-ACTX-Hv1c, and mapped the key functional residues using site-directed mutagenesis. An alanine scan using a panel of 24 mutants provided the first complete map of the bioactive surface of a spider toxin and revealed that the entire J-ACTX-Hv1c pharmacophore is restricted to seven residues that form a bipartite surface patch on one face of the toxin. However, the primary pharmacophore, or hot spot, is formed by just five residues (Arg(8), Pro(9), Tyr(31), and the Cys(13)-Cys(14) vicinal disulfide). The Arg(8)-Tyr(31) diad in J-ACTX-Hv1c superimposes closely on the Lys-(Tyr/Phe) diad that is spatially conserved across a range of structurally dissimilar K+ channel blockers, which leads us to speculate that the J-ACTXs might target an invertebrate K+ channel.