Structure and Function of Integrase

描述（由申请方提供）：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靶标。这些努力目前受到IN结构和IN与DNA底物相互作用方式的不完整知识的限制。因此，所描述的研究的一个主要目标是获得IN的分子结构及其与DNA的相互作用的更详细的了解。目的1将使用X射线晶体学来分析HIV IN结合到设计用于稳定复合物的DNA底物上。将通过NMR光谱和生物化学方法研究C-末端结构域对DNA结合的贡献。第二个目标是开发抑制性HIV IN抗体，在P.I.的实验室，研究蛋白质的构象和功能。目的2将优化条件的结晶复合物，包括全长IN和C-末端定向单克隆Fab，其分析将提供进一步的详细信息IN结构和抑制这种抗体的机制。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，以测试关于C-末端和N-末端定向抗体抑制的特定结构假设。本提案的具体目标基于P.I.的丰富经验和技术专长。和合作者，以及在P.l.的实验室在上一个资助期内。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的病毒酶的新策略。

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.