Structure and Function of Integrase

Integrase的结构和功能

• 批准号: 7560393
• 负责人: ANNA MARIE SKALKA
• 金额: $ 41.94万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7560393
• 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）：综合酶（in）是三种对逆转录病毒复制至关重要的病毒编码酶之一，因此，是制定艾滋病治疗策略的重要目标。尽管我们和其他抗病毒活性抑制剂中的某些HIV-1已被我们和其他抗病毒活性鉴定出来，但由于缺乏有关活动中活性分子结构的关键细节及其与病毒和宿主DNA底物的相互作用，因此进步受到了严重限制。该竞争续约中描述的新方法旨在缩小此差距，从而受益于开发用于研究ASV IN的实验系统。前两个目的解决了DNA相互作用的特定特征：AIM 1将在促进或稳定病毒DNA末端的残基中识别ASV和HIV-1，这是通过使用化学探测，生物物理方法和诱变所需的加工所需的步骤。 AIM 2将使用新的照片交联方法与DNA复合物的分子模型一起绘制IN和病毒和靶DNA之间的特定接触。建模和测试的迭代过程将确定在IN和底物DNA之间的所有相关接触位点。在AIM 3中，将通过动态光散射和小角度X射线散射方法来分析蛋白质和DNA复合物中的ASV中，以确定其在溶液中的组成和构象。化学诱捕方法将用于制备共价连接的，与生物学相关的DNA复合物进行结构分析。这些分析的结果将确认这些复合物中域和底物DNA的化学计量和空间取向，并支持AIM 2的建模过程。在结晶试验中，将测试均质的复合物制剂，目的是在原子分辨率下获取结构数据。关于从这些研究得出的结构和机制的预测将用HIV-1 IN测试。该提案中的实验旨在详细了解HIV-1 IN及其DNA底物之间的分子相互作用。此类信息对于利用这种病毒蛋白作为设计针对艾滋病的新疗法的靶标至关重要。