Substrate N2 atom recognition mechanism in pierisin family DNA-targeting, guanine-specific ADP-ribosyltransferase ScARP
Substrate N2 atom recognition mechanism in pierisin family DNA-targeting, guanine-specific ADP-ribosyltransferase ScARP
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DOI：
10.1074/jbc.ac118.004412
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发表时间：
2018-08
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影响因子：
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通讯作者：
Toru Yoshida;H. Tsuge
中科院分区：
文献类型：
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作者：
Toru Yoshida;H. Tsuge
来自天蓝色链霉菌（Streptomyces coelicolor）的ScARP属于靶向DNA的ADP -核糖基转移酶（ARTs）的穿孔素（pierisin）家族。这些酶将DNA中鸟嘌呤残基的N2氨基进行ADP -核糖基化，生成N2 -（ADP -核糖 - 1 -基）- 2′ -脱氧鸟苷。尽管穿孔素 - 1和斯卡宾（Scabin）的结构最近已被揭示，但由于缺乏底物结合结构，底物识别机制仍知之甚少。在此，我们分别报道了ScARP的无底物结合结构以及ScARP分别与NADH及其GDP底物结合的结构，分辨率分别为1.50 Å和1.57 Å。结合结构显示GDP的鸟嘌呤被夹在NADH的N -核糖和色氨酸 - 159之间。有趣的是，鸟嘌呤的N2和N3分别与谷氨酰胺 - 162的OE1和NE2原子形成氢键。我们直接观察到，包括色氨酸 - 159和谷氨酰胺 - 162在内的ADP -核糖基化毒素转角 - 转角（ARTT）环在靶向DNA的鸟嘌呤特异性ARTs以及靶向蛋白质的ARTs（如C3外酶）的特异性中起关键作用。我们提出ARTT环识别是穿孔素家族中一种常见的底物识别机制。此外，这种复合物结构揭示了由保守结构基序区分的两个亚类之间的相似性和差异：ARTD亚家族中的H - Y - E和ARTC亚家族中的R - S - E。亲电体和亲核体的空间排列相同，为这些ARTs中存在一种共同的反应机制提供了首个证据。ARTC（包括ScARP）利用ARTT环进行底物识别，而ARTD（以Arr为代表）则使用C末端螺旋而非ARTT环。这些观察结果可能有助于为改进ART抑制剂的研究提供信息。
ScARP from the bacterium Streptomyces coelicolor belongs to the pierisin family of DNA-targeting ADP-ribosyltransferases (ARTs). These enzymes ADP-ribosylate the N2 amino groups of guanine residues in DNA to yield N2-(ADP-ribos-1-yl)-2′-deoxyguanosine. Although the structures of pierisin-1 and Scabin were revealed recently, the substrate recognition mechanisms remain poorly understood because of the lack of a substrate-binding structure. Here, we report the apo structure of ScARP and of ScARP bound to NADH and its GDP substrate at 1.50 and 1.57 Å resolutions, respectively. The bound structure revealed that the guanine of GDP is trapped between N-ribose of NADH and Trp-159. Interestingly, N2 and N3 of guanine formed hydrogen bonds with the OE1 and NE2 atoms of Gln-162, respectively. We directly observed that the ADP-ribosylating toxin turn-turn (ARTT)-loop, including Trp-159 and Gln-162, plays a key role in the specificity of DNA-targeting, guanine-specific ARTs as well as protein-targeting ARTs such as the C3 exoenzyme. We propose that the ARTT-loop recognition is a common substrate-recognition mechanism in the pierisin family. Furthermore, this complex structure sheds light on similarities and differences among two subclasses that are distinguished by conserved structural motifs: H-Y-E in the ARTD subfamily and R-S-E in the ARTC subfamily. The spatial arrangements of the electrophile and nucleophile were the same, providing the first evidence for a common reaction mechanism in these ARTs. ARTC (including ScARP) uses the ARTT-loop for substrate recognition, whereas ARTD (represented by Arr) uses the C-terminal helix instead of the ARTT-loop. These observations could help inform efforts to improve ART inhibitors.