Structure and Function of Integrase

Integrase的结构和功能

• 批准号: 6766789
• 负责人: ANNA MARIE SKALKA
• 金额: $ 48.72万
• 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 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6766789
• 关键词: DNA binding protein; DNA footprinting; X ray crystallography; avian sarcoma virus; enzyme activity; enzyme structure; human immunodeficiency virus 1; immunoglobulin structure; integrase; monoclonal antibody; nuclear magnetic resonance spectroscopy; protein structure function; site directed mutagenesis; structural biology; virus DNA

DESCRIPTION (provided by applicant): The retroviral enzyme, integrase (IN) catalyzes the insertion of newly synthesized viral DNA into the host cell chromatin. As DNA integration is required for efficient viral replication, IN is an attractive target for rational drug design. Such efforts are currently limited by an incomplete knowledge of IN structure and the manner in which IN interacts with DNA substrates. Thus, one major goal of the research described is to obtain a more detailed understanding of the molecular structure of IN and its interaction with DNA. Aim 1 will use X-ray crystallography to analyze HIV IN bound to DNA substrates designed to stabilize the complexes. The contribution of the C-terminal domain to DNA binding will be investigated by NMR spectroscopy and biochemical methods. A second goal is to exploit inhibitory HIV IN antibodies, developed and characterized in the P.l.'s laboratory, to investigate protein conformation and function. Aim 2 will optimize conditions for crystallization of a complex that includes full-length IN and a C-terminal-directed monoclonal Fab; its analysis will provide further details of IN structure and the mechanism of inhibition by this antibody. In addition, amino acid substitutions will be introduced into HIV-1 IN to test specific structure-based hypotheses concerning inhibition by the C-terminal- as well as an N-terminal-directed antibody, based on molecular models of IN-Fab complexes. The Specific Aims of this proposal build upon the extensive experience and technical expertise of the P.I. and collaborators, and the discoveries made in the P.l.'s laboratory during the last funding period. The knowledge gained will increase understanding of the structure and function of IN, and suggest new strategies to inhibit this essential viral enzyme.

描述（由申请人提供）：逆转录酶，整合酶（IN）催化新合成的病毒DNA插入宿主细胞染色质。由于有效的病毒复制需要DNA整合，因此IN是合理设计药物的一个有吸引力的靶点。这种努力目前受到对IN结构和IN与DNA底物相互作用方式的不完全了解的限制。因此，该研究的一个主要目标是更详细地了解IN的分子结构及其与DNA的相互作用。目的1将使用x射线晶体学分析HIV IN结合到旨在稳定复合物的DNA底物上。c端结构域对DNA结合的贡献将通过核磁共振波谱和生化方法进行研究。第二个目标是开发抑制HIV IN抗体，在p.l.开发和表征。来研究蛋白质的构象和功能。目的2将优化包括全长IN和c端定向单克隆Fab的复合物的结晶条件；其分析将进一步提供IN结构的细节和该抗体的抑制机制。此外，基于In - fab复合物的分子模型，将氨基酸取代引入HIV-1 In，以测试基于c端和n端定向抗体抑制的特定结构假设。该提案的具体目标建立在私家侦探和合作者的丰富经验和技术专长以及在私家侦探中取得的发现之上。在上一个资助期内，他在美国的实验室工作。所获得的知识将增加对IN的结构和功能的理解，并提出抑制这种必需病毒酶的新策略。