Structure and Function of Integrase

描述（由申请人提供）：整合酶（IN）是三种病毒编码酶之一，对逆转录病毒复制至关重要，因此是制定艾滋病治疗策略的重要靶标。尽管我们和其他人已经发现了一些具有抗病毒活性的HIV-1 IN抑制剂，但由于缺乏有关活性IN复合物的分子结构及其与病毒和宿主DNA底物相互作用的关键细节，进展受到严重限制。这种竞争性更新中描述的新方法旨在缩小这一差距，受益于为研究ASV in而开发的实验系统。前两个目标针对IN-DNA相互作用的特定特征：Aim 1将通过化学探测、生物物理方法和诱变，鉴定ASV和HIV-1的IN残基，这些残基促进或稳定病毒DNA末端解配对，这是处理所需的一个步骤。目的2将利用新的光交联方法和IN-DNA复合物的分子模型绘制IN与病毒和靶DNA之间的特定接触。建模和测试的迭代过程将确定IN和底物dna之间的所有相关接触位点。在Aim 3中，ASV In蛋白和In - dna复合物将通过动态光散射和小角x射线散射方法进行分析，以确定它们在溶液中的组成和构象。化学捕获方法将用于制备共价连接，生物相关的IN-DNA复合物进行结构分析。这些分析的结果将证实这些复合物中IN结构域和底物dna的化学计量学和空间取向，并支持Aim 2的建模过程。均相复合物将在结晶试验中进行测试，目的是获得原子分辨率的结构数据。从这些研究中得出的有关结构和机制的预测将用HIV-1 IN进行测试。本提案中的实验旨在详细了解HIV-1 in与其DNA底物之间的分子相互作用。这些信息对于利用这种病毒蛋白作为设计抗艾滋病新疗法的靶点至关重要。

DESCRIPTION (provided by applicant): Integrase (IN) is one of three viral-encoded enzymes that are essential for retroviral replication and, therefore, an important target for the development of strategies for the treatment of AIDS. Although some HIV-1 IN inhibitors with antiviral activity have been identified by us and others, progress has been seriously limited by a lack of critical details concerning the molecular structure of the active IN complex and its interactions with viral and host DNA substrates. The new approaches described in this competing renewal are designed to close this gap, benefiting from experimental systems developed to study ASV IN. The first two Aims address specific features of IN-DNA interactions: Aim 1 will identify ASV and HIV-1 IN residues that promote or stabilize viral DNA end unpairing, a step required for processing, by using chemical probing, biophysical methods, and mutagenesis. Aim 2 will map specific contacts between IN and viral and target DNA using new photo crosslinking methods together with molecular models of IN-DNA complexes. An iterative process of modeling and testing will identify all relevant contact sites between IN and substrate DNAs. In Aim 3 ASV IN proteins and IN-DNA complexes will be analyzed by dynamic light scattering and small angle X-ray scattering methods to determine their composition and conformation in solution. Chemical trapping methods will be used to prepare covalently linked, biologically-relevant IN-DNA complexes for structural analysis. The results of these analyses will confirm the stoichiometry and spatial orientation of IN domains and substrate DNAs in these complexes and support the modeling process of Aim 2. Homogeneous complex preparations will be tested in crystallization trials with the goal of acquiring structural data at atomic resolution. Predictions concerning structure and mechanism derived from these studies will be tested with HIV-1 IN. Experiments in this proposal are designed to provide a detailed understanding of the molecular interactions between HIV-1 IN and its DNA substrates. Such information is crucial for exploiting this viral protein as a target for the design of new therapies against AIDS.