Structure and Function of Integrase

描述(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DNA整合是病毒有效复制所必需的，因此IN是合理药物设计的一个有吸引力的靶点。目前这种努力受到对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，所描述的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。AIM 1将使用X射线结晶学来分析与DNA底物结合的HIV IN，该底物设计用于稳定复合体。C-末端结构域对DNA结合的贡献将通过核磁共振光谱和生物化学方法进行研究。第二个目标是利用中国人民解放军S实验室开发并鉴定的抑制性HIVIN抗体，以研究蛋白质的构象和功能。AIM 2将优化包括全长IN和C末端导向的单抗Fab的复合体的结晶条件；其分析将提供IN结构和该抗体抑制机制的更多细节。此外，将在HIV-1IN中引入氨基酸取代，以基于IN-Fab复合体的分子模型，测试关于C末端和N末端定向抗体抑制作用的特定结构假说。这项建议的具体目标是建立在P.I.及其合作者的广泛经验和技术专长，以及在上一次资助期间在P.L.S实验室所取得的发现的基础上的。所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。

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.