Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin.
Crystal structure and structure-based mutagenesis of actin-specific ADP-ribosylating toxin CPILE-a as novel enterotoxin.
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DOI：
10.1371/journal.pone.0171278
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Tsuge H
中科院分区：
文献类型：
--
作者：
Toniti W;Yoshida T;Tsurumura T;Irikura D;Monma C;Kamata Y;Tsuge H
日本报告了不寻常的食物中毒爆发，根据流行病学信息和分离株的指纹分析，强烈怀疑产气荚膜梭菌是原因。分离菌株缺乏典型的C.产气荚膜梭菌肠毒素（CPE），但分泌一种新的肠毒素，由两种成分组成：C.产气荚膜杆菌样肠毒素-a（CPILE-a），其充当酶促ADP-核糖基转移酶，和CPILE-b，膜结合组分。本文报道了apo-CPILE-a、NAD+-CPILE-a和NADH-CPILE-a的晶体结构。虽然CPILE-a结构与已知的NAD+ iota毒素-a（Ia）具有高度相似性，但它具有两个不同于Ia的来自G262-S269和E402-K408的超长突出环。基于Ia-肌动蛋白复合物的结构，我们重点研究了包括两个突出环（PT）的肌动蛋白结合界面区域（I-V），并研究了这些区域的突变如何影响CPILE-a的ADP核糖基化活性。虽然已经对Ia的肌动蛋白结合位点进行了一些定点突变研究，但在本研究中，对CPILE-a和Ia中的α-和β/γ-肌动蛋白进行了突变研究。有趣的是，CPILE-a ADP-核糖基化α-和β/γ-肌动蛋白，但其对β/γ-肌动蛋白的敏感性是α-肌动蛋白的36%。我们的结果与仅C2-IADP-核糖基化β/γ-actin的结果相反。我们还表明，PT-I和CPILE-a中的两个凹凸相互作用对于肌动蛋白结合是重要的。目前的研究是第一次详细分析Ia和CPILE-a对α-和β/γ-肌动蛋白的肌动蛋白结合区的定点突变。
Unusual outbreaks of food poisoning in Japan were reported in which Clostridium perfringens was strongly suspected to be the cause based on epidemiological information and fingerprinting of isolates. The isolated strains lack the typical C. perfringens enterotoxin (CPE) but secrete a new enterotoxin consisting of two components: C. perfringens iota-like enterotoxin-a (CPILE-a), which acts as an enzymatic ADP-ribosyltransferase, and CPILE-b, a membrane binding component. Here we present the crystal structures of apo-CPILE-a, NAD+-CPILE-a and NADH-CPILE-a. Though CPILE-a structure has high similarity with known iota toxin-a (Ia) with NAD+, it possesses two extra-long protruding loops from G262-S269 and E402-K408 that are distinct from Ia. Based on the Ia–actin complex structure, we focused on actin-binding interface regions (I-V) including two protruding loops (PT) and examined how mutations in these regions affect the ADP-ribosylation activity of CPILE-a. Though some site-directed mutagenesis studies have already been conducted on the actin binding site of Ia, in the present study, mutagenesis studies were conducted against both α- and β/γ-actin in CPILE-a and Ia. Interestingly, CPILE-a ADP-ribosylates both α- and β/γ-actin, but its sensitivity towards β/γ-actin is 36% compared with α-actin. Our results contrast to that only C2-I ADP-ribosylates β/γ-actin. We also showed that PT-I and two convex-concave interactions in CPILE-a are important for actin binding. The current study is the first detailed analysis of site-directed mutagenesis in the actin binding region of Ia and CPILE-a against both α- and β/γ-actin.