The local dinucleotide preference of APOBEC3G can be altered from 5'-CC to 5'-TC by a single amino acid substitution.
The local dinucleotide preference of APOBEC3G can be altered from 5'-CC to 5'-TC by a single amino acid substitution.
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DOI：
10.1016/j.jmb.2013.07.040
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发表时间：
2013-11-15
影响因子：
5.6
通讯作者：
Harris, Reuben S.
中科院分区：
文献类型：
--
作者：
Rathore, Anurag;Carpenter, Michael A.;Demir, Oeziem;Ikeda, Terumasa;Li, Ming;Shaban, Nadine M.;Law, Emily K.;Anokhin, Dmitry;Brown, William L.;Amaro, Rommie E.;Harris, Reuben S.
APOBEC 3A和APOBEC 3G是DNA胞嘧啶脱氨酶，分别在外源DNA和逆转录病毒限制中具有生物学功能。在单链DNA底物中，APOBEC 3A对胞嘧啶具有内在偏好，而在胞嘧啶之前是胸腺嘧啶（5′-TC），而APOBEC 3G更喜欢靶胞嘧啶之前是另一个胞嘧啶（5′-CC）。为了确定负责这些强二核苷酸偏好的氨基酸，我们分析了一系列嵌合体，其中APOBEC 3G的推定DNA结合环区域被APOBEC 3A的相应区域取代。环3替换增强APOBEC 3G催化活性，但不改变其固有的5′-CC二核苷酸底物偏好。环7的替换导致APOBEC 3G变成APOBEC 3A样，并强烈偏好5′-TC底物。同时环3/7替换导致高度活跃的APOBEC 3G变体，其也偏好5′-TC二核苷酸。单氨基酸交换显示D317作为二核苷酸底物特异性的关键决定因素。多拷贝明确溶剂化的全原子分子动力学模拟提出了一个模型，其中D317作为一个螺旋帽残基通过限制环7的流动性，形成一个新的结合口袋，有利地容纳胞嘧啶。所有具有催化活性的APOBEC 3G变体，无论二核苷酸偏好如何，都保留了HIV-1限制活性。这些数据支持一个模型，其中环7区域控制脱氨基作用的局部二核苷酸底物的选择，但不太可能是将这些酶导向生物底物（如HIV-1 cDNA）的更高水平靶向机制的一部分。
APOBEC3A and APOBEC3G are DNA cytosine deaminases with biological functions in foreign DNA and retrovirus restriction, respectively. APOBEC3A has an intrinsic preference for cytosine preceded by thymine (5′-TC) in single-stranded DNA substrates, whereas APOBEC3G prefers the target cytosine to be preceded by another cytosine (5′-CC). To determine the amino acids responsible for these strong dinucleotide preferences, we analyzed a series of chimeras in which putative DNA binding loop regions of APOBEC3G were replaced with the corresponding regions from APOBEC3A. Loop 3 replacement enhanced APOBEC3G catalytic activity but did not alter its intrinsic 5′-CC dinucleotide substrate preference. Loop 7 replacement caused APOBEC3G to become APOBEC3A-like and strongly prefer 5′-TC substrates. Simultaneous loop 3/7 replacement resulted in a hyperactive APOBEC3G variant that also preferred 5′-TC dinucleotides. Single amino acid exchanges revealed D317 as a critical determinant of dinucleotide substrate specificity. Multi-copy explicitly solvated all-atom molecular dynamics simulations suggested a model in which D317 acts as a helix-capping residue by constraining the mobility of loop 7, forming a novel binding pocket that favorably accommodates cytosine. All catalytically active APOBEC3G variants, regardless of dinucleotide preference, retained HIV-1 restriction activity. These data support a model in which the loop 7 region governs the selection of local dinucleotide substrates for deamination but is unlikely to be part of the higher level targeting mechanisms that direct these enzymes to biological substrates such as HIV-1 cDNA.
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影响因子：
5.5
作者：
Goetz, Andreas W.;Williamson, Mark J.;Xu, Dong;Poole, Duncan;Le Grand, Scott;Walker, Ross C.
通讯作者：
Walker, Ross C.
影响因子：
16.6
作者：
通讯作者：
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影响因子：
64.5
作者：
Harris, RS;Bishop, KN;Malim, MH
通讯作者：
Malim, MH
影响因子：
16
作者：
Harris, RS;Petersen-Mahrt, SK;Neuberger, MS
通讯作者：
Neuberger, MS
影响因子：
3.7
作者：
Browne, Edward P.;Allers, Carolina;Landau, Nathaniel R.
通讯作者：
Landau, Nathaniel R.