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必不可少的一步。这使得整合酶成为一个有吸引力的药物靶点，而抗整合酶药物正在美国的临床试验中。合理的药物设计得益于可视化的三维酶结构，许多HIV-1整合酶催化核心区域的结构已经得到解决。虽然两个结构域n端/核心和核心/c端结构最近才被解决，但没有完整的整合酶结构，这在很大程度上可能是由于蛋白质溶解度差。事实上，所有先前的晶体在催化核心区域都含有一个或多个增溶性突变，以F185K为典型。在细胞中，整合酶可能与正常的人类蛋白质相互作用，整合酶与最近发现的人晶状体上皮衍生生长因子（LEDGF）之间的相互作用形成了这种应用的基础。我们将利用LEDGF蛋白提高HIV-1整合酶的溶解度，并结晶LEDGF整合酶复合物。基于其在生理相关缓冲条件下的高溶解度限制，我们得出结论，与整合酶不同，LEDGF在溶液中表现良好。初步结果表明，纯化的LEDGF蛋白显著提高了纯化的HIV-1整合酶在生理相关缓冲条件下的溶解度，这为该提议的科学依据做出了定义。通过有限的蛋白水解对LEDGF结构的进一步探测发现了一个胰蛋白酶抗性亚结构域，这对整合酶结合和溶解活性很重要。我们建议利用核磁共振光谱技术解析LEDGF整合酶结合亚域的结构，并将该亚域或全长LEDGF与HIV-1整合酶结合在一起进行结晶。这些实验的结果将定义整合酶的一个重要细胞相互作用物的区域结构，并为解决完整的HIV-1酶的三维结构奠定基础。

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.