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首页> 外文期刊>Journal of Molecular Biology >Discovery and structural characterization of a new inhibitor series of HIV-1 nucleocapsid function: NMR solution structure determination of a ternary complex involving a 2:1 inhibitor/NC stoichiometry
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Discovery and structural characterization of a new inhibitor series of HIV-1 nucleocapsid function: NMR solution structure determination of a ternary complex involving a 2:1 inhibitor/NC stoichiometry

机译：发现和一系列HIV-1核衣壳功能的新抑制剂的结构表征：涉及2：1抑制剂/ NC化学计量的三元复合物的NMR溶液结构测定

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The nucleocapsid (NC) protein is an essential factor with multiple functions within the human immunodeficiency virus type 1 (HIV-1) replication cycle. In this study, we describe the discovery of a novel series of inhibitors that targets HIV-1 NC protein by blocking its interaction with nucleic acids. This series was identified using a previously described capsid (CA) assembly assay, employing a recombinant HIV-1 CA-NC protein and immobilized TG-rich deoxyoligonucleotides. Using visible absorption spectroscopy, we were able to demonstrate that this new inhibitor series binds specifically and reversibly to the NC with a peculiar 2:1 stoichiometry. A fluorescence-polarization-based binding assay was also developed in order to monitor the inhibitory activities of this series of inhibitors. To better characterize the structural aspect of inhibitor binding onto NC, we performed NMR studies using unlabeled and 13C,15N-double-labeled NC(1-55) protein constructs. This allowed the determination of the solution structure of a ternary complex characterized by two inhibitor molecules binding to the two zinc knuckles of the NC protein. To the best of our knowledge, this represents the first report of a high-resolution structure of a small-molecule inhibitor bound to NC, demonstrating sub-micromolar potency and moderate antiviral potency with one analogue of the series. This structure was compared with available NC/oligonucleotide complex structures and further underlined the high flexibility of the NC protein, allowing it to adopt many conformations in order to bind its different oligonucleotideucleomimetic targets. In addition, analysis of the interaction details between the inhibitor molecules and NC demonstrated how this novel inhibitor series is mimicking the guanosine nucleobases found in many reported complex structures.
机译：核衣壳（NC）蛋白是人类免疫缺陷病毒1型（HIV-1）复制周期内具有多种功能的必需因子。在这项研究中，我们描述了通过阻断HIV-1 NC蛋白与核酸的相互作用而靶向HIV-1 NC蛋白的一系列新型抑制剂的发现。使用重组HIV-1 CA-NC蛋白和固定化的富含TG的脱氧寡核苷酸，使用先前描述的衣壳（CA）组装测定法鉴定了该系列。使用可见吸收光谱，我们能够证明该新型抑制剂系列以独特的2：1化学计量比可逆地与NC特异性结合。还开发了一种基于荧光偏振的结合测定法，以监测该系列抑制剂的抑制活性。为了更好地表征抑制剂与NC结合的结构方面，我们使用未标记和13C，15N-双标记NC（1-55）蛋白构建体进行了NMR研究。这样就可以确定三元复合物的溶液结构，其特征是两个抑制剂分子与NC蛋白的两个锌指结合。据我们所知，这代表了与NC结合的小分子抑制剂的高分辨率结构的首次报道，证明了该系列的一个类似物具有亚微摩尔效价和中等抗病毒效价。将该结构与可用的NC /寡核苷酸复合物结构进行了比较，并进一步强调了NC蛋白的高度灵活性，使其可以采用许多构象以结合其不同的寡核苷酸/拟核苷酸靶标。此外，对抑制剂分子与NC之间相互作用细节的分析表明，该新型抑制剂系列是如何模仿在许多报道的复杂结构中发现的鸟苷核苷碱基的。

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