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中。由于DNA积分是有效的病毒复制所必需的，因此IN是合理药物设计的有吸引力的目标。目前，这种努力受到结构不完整的知识以及与DNA底物相互作用的方式的限制。因此，所描述的研究的一个主要目标是对IN的分子结构及其与DNA的相互作用获得更详细的了解。 AIM 1将使用X射线晶体学来分析旨在稳定复合物的DNA底物的HIV。 C末端结构域对DNA结合的贡献将通过NMR光谱和生化方法研究。第二个目标是利用P.L.实验室中开发和表征的抗体中的抑制性HIV，以研究蛋白质构象和功能。 AIM 2将优化包括全长中和C末端指导的单克隆晶圆剂的复合物结晶条件；它的分析将提供进一步的结构细节和该抗体抑制的机理。此外，将基于投资组内复合物的分子模型，将氨基酸取代引入HIV-1中，以测试C末端 - 和N末端定向的抗体抑制的特定基于结构的假设。该提案的具体目的是基于P.I.的广泛经验和技术专长。和合作者以及在最后一个资金期间在P.L.实验室中提出的发现。获得的知识将增加对IN结构和功能的理解，并提出抑制这种基本病毒酶的新策略。