Crystal Structure of a Soluble APOBEC3G Variant Suggests ssDNA to Bind in a Channel that Extends between the Two Domains.
Crystal Structure of a Soluble APOBEC3G Variant Suggests ssDNA to Bind in a Channel that Extends between the Two Domains.
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DOI：
10.1016/j.jmb.2020.10.020
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发表时间：
2020-11-20
影响因子：
5.6
通讯作者：
Matsuo H
中科院分区：
文献类型：
--
作者：
Maiti A;Myint W;Delviks-Frankenberry KA;Hou S;Kanai T;Balachandran V;Sierra Rodriguez C;Tripathi R;Kurt Yilmaz N;Pathak VK;Schiffer CA;Matsuo H
APOBEC3G (A3G) 是一种单链 DNA (ssDNA) 胞嘧啶脱氨酶，可以通过突变病毒基因组来限制 HIV-1 感染。 A3G 由通过短接头连接的非催化 N 端结构域 (NTD) 和催化 C 端结构域 (CTD) 组成。 CTD 催化胞嘧啶脱氨基作用，而 NTD 被认为可以为 ssDNA 提供额外的亲和力。两个 A3G 域的结构均已单独求解；然而，全长的 A3G 结构一直具有挑战性。最近，全长恒河猴 A3G 变体的晶体结构得到解决，这表明二聚化机制和 RNA 结合表面，而二聚化似乎会损害催化活性。我们确定了人 A3G 可溶性变体 (sA3G) 2.5Å 的晶体结构，并根据这些数据生成了野生型 A3G 的模型结构。该模型证明 NTD 相对于 CTD 沿分子主轴旋转 90°，该方向形成连接到 CTD 催化位点的带正电通道，由 NTD Loop-1 和 CTD Loop-3 组成。基于结构的突变、体外脱氨基和 DNA 结合测定以及 HIV-1 限制性测定确定了位于 NTD Loop-1 中的 R24，对于与 ssDNA 的关键相互作用至关重要。此外，sA3G 显示在催化 Zn2+ 附近结合脱氧胞苷二核苷酸，但不在催化位置，其中脱氧胞苷与 CTD 环 1 和环 7 残基之间的相互作用不同于与底物形成的相互作用。这些新的相互作用提出了一种机制，可以解释为什么 A3G 在进行性脱氨基作用中表现出 3' 至 5' 方向的偏好。
APOBEC3G (A3G) is a single-stranded DNA (ssDNA) cytosine deaminase that can restrict HIV-1 infection by mutating the viral genome. A3G consists of a non-catalytic N-terminal domain (NTD) and a catalytic C-terminal domain (CTD) connected by a short linker. While the CTD catalyzes cytosine deamination, the NTD is believed to provide additional affinity for ssDNA. Structures of both A3G domains have been solved individually; however, a full-length A3G structure has been challenging. Recently, crystal structures of full-length rhesus macaque A3G variants were solved which suggested dimerization mechanisms and RNA binding surfaces, whereas the dimerization appeared to compromise catalytic activity. We determined the crystal structure of a soluble variant of human A3G (sA3G) at 2.5Å and from these data generated a model structure of wild-type A3G. This model demonstrated that the NTD was rotated 90° relative to the CTD along the major axis of the molecule, an orientation that forms a positively charged channel connected to the CTD catalytic site, consisting of NTD loop-1 and CTD loop-3. Structure-based mutations, in vitro deamination and DNA binding assays, and HIV-1 restriction assays identify R24, located in the NTD loop-1, as essential to a critical interaction with ssDNA. Furthermore, sA3G was shown to bind a deoxy-cytidine dinucleotide near the catalytic Zn2+, yet not in the catalytic position, where the interactions between deoxy-cytidines and CTD loop-1 and loop-7 residues were different from those formed with substrate. These new interactions suggest a mechanism explaining why A3G exhibits a 3' to 5' directional preference in processive deamination.
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影响因子：
5.6
作者：
Desimmie, Belete A.;Delviks-Frankenberrry, Krista A.;Burdick, Ryan C.;Qi, DongFei;Izumi, Taisuke;Pathak, Vinay K.
通讯作者：
Pathak, Vinay K.
影响因子：
16.6
作者：
Bohn JA;Thummar K;York A;Raymond A;Brown WC;Bieniasz PD;Hatziioannou T;Smith JL
通讯作者：
Smith JL
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
4.8
作者：
Dang, Ying;Siew, Lai Mun;Zheng, Yong-Hui
通讯作者：
Zheng, Yong-Hui
影响因子：
4.8
作者：
Chelico, Linda;Prochnow, Courtney;Goodman, Myron F.
通讯作者：
Goodman, Myron F.