LEDGF-Integrase Structural Biology

描述（由申请人提供）：HIV-1 cDNA整合到细胞染色体中是病毒复制周期中的重要步骤。这使得整合酶成为一个有吸引力的药物靶点，抗整合酶药物正在美国进行临床试验。合理的药物设计受益于三维酶结构的可视化，并且HIV-1整合酶催化核心结构域的许多结构已经被解决。虽然最近解决了两个结构域N-末端/核心和核心/C-末端结构，但没有完整整合酶的结构，这在很大程度上可能是由于蛋白质溶解度差。事实上，所有以前的晶体都在催化核心结构域中含有一个或多个增溶突变，以F185 K为代表。在细胞中，整合酶可能与正常的人类蛋白质相互作用，并且整合酶与最近鉴定的人透镜上皮衍生生长因子（LEDGF）之间的相互作用形成了这种应用的基础。我们将使用LEDGF蛋白来提高HIV-1整合酶的溶解度，并将使LEDGF-整合酶复合物结晶。基于其在生理相关缓冲液条件下的高溶解度极限，我们得出结论，与整合酶不同，LEDGF在溶液中表现良好。该提议的科学基础由初步结果定义，初步结果表明纯化的LEDGF蛋白显著改善了纯化的HIV-1整合酶在生理相关缓冲液条件下的溶解度。通过有限的蛋白水解对LEDGF结构的额外探测确定了对整合酶结合和溶解活性重要的胰蛋白酶抗性亚结构域。我们建议通过NMR光谱解析LEDGF的整合酶结合亚结构域的结构，并使与HIV-1整合酶复合的亚结构域或全长LEDGF结晶。这些实验的结果将定义整合酶的重要细胞相互作用物的区域的结构，以及形成解决完整HIV-1酶的三维结构的基础。

DESCRIPTION (provided by applicant): Integration of HIV-1 cDNA into a cell chromosome is an essential step in the virus replication cycle. This makes the integrase an attractive drug target, and anti-integrase drugs are in US clinical trials. Rational drug design benefits from visualizing three-dimensional enzyme structures, and numerous structures of the HIV-1 integrase catalytic core domain have been solved. Although two domain N-terminal/core and core/C-terminal structures were more recently solved, there is no structure for the intact integrase, which in large part may be due to poor protein solubility. Indeed, all previous crystals contained one or more solubilizing mutations in the catalytic core domain, typified by F185K. In cells, integrase is likely to interact with normal human proteins, and the interaction between integrase and the recently identified human lens epithelium-derived growth factor (LEDGF) forms the basis for this application. We will use LEDGF protein to improve the solubility of HIV-1 integrase, and will crystallize LEDGF-integrase complexes. Based on its high solubility limit in physiologically-relevant buffer conditions, we conclude that unlike the integrase, LEDGF is well behaved in solution. The scientific basis for this proposal is defined by preliminary results demonstrating that purified LEDGF protein significantly improved the solubility of purified HIV-1 integrase in physiologically- relevant buffer conditions. Additional probing of the LEDGF structure by limited proteolysis identified a trypsin-resistant subdomain important for integrase binding and solubilization activities. We propose to solve the structure of the integrase binding subdomain of LEDGF by NMR spectroscopy, and crystallize the subdomain or full-length LEDGF in complex with HIV-1 integrase. The results of these experiments will define the structure of the region of an important cellular interactor for the integrase, as well as form the basis for solving the three dimensional structure of the intact HIV-1 enzyme.