Department of Biochemistry, Molecular Biology and Biophysics, Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455, USA.
Biology (Basel). 2012 Aug 2;1(2):260-76. doi: 10.3390/biology1020260.
APOBEC3G is the best known of several DNA cytosine deaminases that function to inhibit the replication of parasitic genetic elements including the lentivirus HIV. Several high-resolution structures of the APOBEC3G catalytic domain have been generated, but none reveal how this enzyme binds to substrate single-stranded DNA. Here, we constructed a panel of APOBEC3G amino acid substitution mutants and performed a series of biochemical, genetic, and structural assays to distinguish between "Brim" and "Kink" models for single-strand DNA binding. Each model predicts distinct sets of interactions between surface arginines and negatively charged phosphates in the DNA backbone. Concordant with both models, changing the conserved arginine at position 313 to glutamate abolished both catalytic and restriction activities. In support of the Brim model, arginine to glutamate substitutions at positions 213, 215, and 320 also compromised these APOBEC3G activities. Arginine to glutamate substitutions at Kink model residues 374 and 376 had smaller effects. These observations were supported by A3G catalytic domain-ssDNA chemical shift perturbation experiments. The overall data set is most consistent with the Brim model for single-stranded DNA binding by APOBEC3G.
APOBEC3G 是几种 DNA 胞嘧啶脱氨酶中最著名的一种，其功能是抑制包括慢病毒 HIV 在内的寄生遗传元件的复制。已经生成了几种 APOBEC3G 催化结构域的高分辨率结构，但没有一种揭示了该酶如何与底物单链 DNA 结合。在这里，我们构建了一组 APOBEC3G 氨基酸取代突变体，并进行了一系列生化、遗传和结构测定，以区分单链 DNA 结合的“Brim”和“Kink”模型。每个模型都预测了表面精氨酸与 DNA 骨架中带负电荷的磷酸之间的不同的相互作用。与两种模型一致，将位置 313 的保守精氨酸突变为谷氨酸，既消除了催化活性，也消除了限制活性。支持 Brim 模型，位置 213、215 和 320 的精氨酸突变为谷氨酸也会损害 APOBEC3G 的这些活性。在 Kink 模型残基 374 和 376 处的精氨酸突变为谷氨酸的影响较小。这些观察结果得到了 A3G 催化结构域-ssDNA 化学位移扰动实验的支持。总的数据集最符合 APOBEC3G 与单链 DNA 结合的 Brim 模型。
