Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, United States of America.
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America.
PLoS One. 2018 Mar 29;13(3):e0195048. doi: 10.1371/journal.pone.0195048. eCollection 2018.
Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (A3) proteins are a family of cytidine deaminases that catalyze the conversion of deoxycytidine (dC) to deoxyuridine (dU) in single-stranded DNA (ssDNA). A3 proteins act in the innate immune response to viral infection by mutating the viral ssDNA. One of the most well-studied human A3 family members is A3G, which is a potent inhibitor of HIV-1. Each A3 protein prefers a specific substrate sequence for catalysis-for example, A3G deaminates the third dC in the CCCA sequence motif. However, the interaction between A3G and ssDNA is difficult to characterize due to poor solution behavior of the full-length protein and loss of DNA affinity of the truncated protein. Here, we present a novel DNA-anchoring fusion strategy using the protection of telomeres protein 1 (Pot1) which has nanomolar affinity for ssDNA, with which we captured an A3G-ssDNA interaction. We crystallized a non-preferred adenine in the -1 nucleotide-binding pocket of A3G. The structure reveals a unique conformation of the catalytic site loops that sheds light onto how the enzyme scans substrate in the -1 pocket. Furthermore, our biochemistry and virology studies provide evidence that the nucleotide-binding pockets on A3G influence each other in selecting the preferred DNA substrate. Together, the results provide insights into the mechanism by which A3G selects and deaminates its preferred substrates and help define how A3 proteins are tailored to recognize specific DNA sequences. This knowledge contributes to a better understanding of the mechanism of DNA substrate selection by A3G, as well as A3G antiviral activity against HIV-1.
人载脂蛋白 B mRNA 编辑酶催化多肽样 3(A3)蛋白是一类胞嘧啶脱氨酶,可催化单链 DNA(ssDNA)中脱氧胞苷(dC)转化为脱氧尿苷(dU)。A3 蛋白通过突变病毒 ssDNA 来参与先天免疫反应以抵抗病毒感染。研究最为深入的人类 A3 家族成员之一是 A3G,它是 HIV-1 的强效抑制剂。每个 A3 蛋白都偏爱特定的催化底物序列,例如,A3G 使 CCCA 序列基序中的第三个 dC 脱氨。然而,由于全长蛋白的溶液行为不佳以及截短蛋白 DNA 亲和力的丧失,A3G 与 ssDNA 之间的相互作用难以表征。在这里,我们提出了一种使用端粒保护蛋白 1(Pot1)的新型 DNA 锚定融合策略,Pot1 对 ssDNA 的亲和力为纳摩尔级,我们利用该策略捕获了 A3G-ssDNA 相互作用。我们对 A3G 中 -1 核苷酸结合口袋中的非优先腺嘌呤进行了结晶。该结构揭示了催化位点环的独特构象,阐明了该酶如何在 -1 口袋中扫描底物。此外,我们的生物化学和病毒学研究提供了证据,表明 A3G 上的核苷酸结合口袋在选择首选 DNA 底物时相互影响。总的来说,这些结果提供了对 A3G 选择和脱氨其首选底物的机制的深入了解,并有助于确定 A3 蛋白如何适应识别特定的 DNA 序列。这些知识有助于更好地理解 A3G 对 DNA 底物选择的机制以及 A3G 对 HIV-1 的抗病毒活性。