Structure and Function of Integrase

Integrase的结构和功能

• 批准号: 6589862
• 负责人: ANNA MARIE SKALKA
• 金额: $ 40.34万
• 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/6589862
• 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）：逆转录病毒酶整合酶（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，IN 是合理药物设计的一个有吸引力的目标。目前，由于对 IN 结构以及 IN 与 DNA 底物相互作用的方式了解不完整，此类努力受到限制。因此，所述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。目标 1 将使用 X 射线晶体学来分析与旨在稳定复合物的 DNA 底物结合的 HIV IN。 C 末端结构域对 DNA 结合的贡献将通过 NMR 光谱和生化方法进行研究。第二个目标是利用 P.l. 实验室开发和表征的抑制性 HIV IN 抗体来研究蛋白质构象和功能。目标 2 将优化包含全长 IN 和 C 端定向单克隆 Fab 的复合物的结晶条件；其分析将提供 IN 结构和该抗体抑制机制的进一步细节。此外，将在 HIV-1 IN 中引入氨基酸取代，以基于 IN-Fab 复合物的分子模型，测试有关 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 P.I. 的丰富经验和技术专长之上。和合作者，以及 P.l. 实验室在上一个资助期间取得的发现。获得的知识将增加对IN结构和功能的理解，并提出抑制这种重要病毒酶的新策略。