Structure and Function of Integrase

Integrase的结构和功能

• 批准号: 8072933
• 负责人: ANNA MARIE SKALKA
• 金额: $ 1.07万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-24 至 2010-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8072933
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Address; Affect; Amino Acid Substitution; Amino Acids; Antiviral Agents; Avian Sarcoma Viruses; Binding; Chemicals; Complement; Complex; Computers; Cross-Linking Reagents; Crystallization; DNA; DNA Integration; Data; Development; Disulfides; Enzymes; Funding; Goals; HIV; HIV-1; HIV-1 integrase; Integrase; Integrase Inhibitors; Knowledge; Laboratories; Link; Maps; Measurement; Measures; Methodology; Methods; Modeling; Molecular Conformation; Molecular Models; Molecular Structure; Mutagenesis; Nature; Nucleotides; Positioning Attribute; Preparation; Process; Proteins; Reaction; Reagent; Resolution; Roentgen Rays; Site; Solutions; Space Perception; Structure; Substrate Domain; System; Testing; Viral; Viral Proteins; Work; base; crosslink; design; flexibility; inhibitor/antagonist; light scattering; molecular modeling; novel strategies; research study; small molecule; stoichiometry; treatment strategy; viral DNA

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.

描述（由申请人提供）：整合酶（IN）是逆转录病毒复制所必需的三种病毒编码酶之一，因此是开发艾滋病治疗策略的重要靶标。虽然我们和其他人已经鉴定出一些具有抗病毒活性的HIV-1 IN抑制剂，但由于缺乏关于活性IN复合物的分子结构及其与病毒和宿主DNA底物的相互作用的关键细节，进展受到严重限制。在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。前两个目标解决了IN-DNA相互作用的具体特征：目标1将通过使用化学探测、生物物理方法和诱变来鉴定促进或稳定病毒DNA末端解配对的ASV和HIV-1 IN残基，这是处理所需的步骤。目的二是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之间的特异性接触图谱。建模和测试的迭代过程将识别IN和底物DNA之间的所有相关接触位点。在目标3中，将通过动态光散射和小角X射线散射方法分析ASV IN蛋白和IN-DNA复合物，以确定它们在溶液中的组成和构象。将使用化学捕获方法制备共价连接的生物相关IN-DNA复合物，用于结构分析。这些分析的结果将证实IN域和底物DNA在这些复合物中的化学计量和空间取向，并支持目标2的建模过程。将在结晶试验中测试均相复合物制剂，目的是获得原子分辨率的结构数据。将用HIV-1 IN测试来自这些研究的关于结构和机制的预测。在这个建议中的实验旨在提供一个详细的了解HIV-1 IN和它的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艾滋病新疗法的靶点至关重要。