Stabilization of CXCR4 and its complexes for high resolution structural studies

描述（由申请人提供）：通过实验确定G蛋白偶联受体（GPCR）、CXCR 4及其复合物的3-D结构，这是拟议研究的总体目标，对于深入了解其在HIV病毒进入和毒性中的作用至关重要;这些数据的可用性有望促进新治疗策略的开发。尽管CCR 5是负责HIV传播的主要辅助受体，但在感染后期出现CXCR 4-tropic（X4-tropic）病毒与更快的CD 4下降和更快的进展为AIDS相关。CXCR 4也与神经艾滋病有关，引发神经元细胞死亡，并可能导致痴呆症的发作。已知的GPCR的构象灵活性以及来自初步研究的结果表明，CXCR 4受体的稳定化是试图结晶和解析其结构的重要瓶颈。该研究计划的两名PI，一名是化学家，另一名是晶体学家，建议开发新的化学工具，用于HIV辅助受体CXCR 4的结构研究，尝试受体及其复合物的结晶和结构测定。化学工具将被设计和优化，用于稳定，纯化和检测受体，并测量其功能行为。R21研究将有两个目标：（1）开发优化用于CXCR 4结构研究的工具化合物，（2）通过评估其对蛋白质样品生产过程的影响和通过生产CXCR 4的衍射晶体来开发新的配体，去污剂和脂质。R21研究的结果将用于在R33阶段实现以下目标：（3）确定CXCR 4的高分辨率X射线结构，以及（4）评估新分子工具和技术在稳定和结晶CXCR 4与gp 120复合物中的效用。R21研究的成功将通过生产衍射CXCR 4晶体沿着生产新的化合物工具来实现，所述新的化合物工具显著改善受体样品行为（例如，随时间推移改善稳定性，增加Tm）。 公共卫生相关性：CXCR 4沿着其复合物的3D结构的可用性将导致对HIV病毒如何进入细胞的更深入的理解，并将有助于设计新的治疗方法。CXCR 4还参与重要的生理过程，并与许多疾病如癌症有关，其结构的可用性同样具有重要的生物医学意义。

DESCRIPTION (provided by applicant): Experimentally determining the 3-D structure of the G protein-coupled receptor (GPCR), CXCR4 and its complexes, the overall goal of the proposed study, is critical in developing a deeper understanding of its role in HIV viral entry and toxicity; availability of these data is expected to facilitate the development of new therapeutic strategies. Although CCR5 is the primary co-receptor responsible for HIV transmission, emergence of CXCR4-tropic (X4-tropic) viruses later in infection, correlates with a more rapid CD4 decline and a faster progression to AIDS. CXCR4 is also implicated in neuroaids, triggering neuronal cell death and possibly causing onset of dementia. Known conformational flexibility of GPCRs as well as results from preliminary studies have shown that stabilization of the CXCR4 receptor is a significant bottleneck in attempts to crystallize and solve its structures. The two PI's of this research proposal, one a chemist, and the other a crystallographer, propose to develop new chemical tools optimized for structural studies of the HIV co-receptor CXCR4, attempting crystallization and structural determination of the receptors and its complexes. Chemical tools will be designed and optimized for stabilizing, purifying, and detecting the receptor and for measuring its functional behavior. R21 studies will have two aims: (1) Develop tool compounds optimized for structural studies of CXCR4 and (2) Validate new ligands, detergents, and lipids by assessing their impact on protein sample production processes and by producing diffracting crystals of CXCR4. Results from R21 studies will be used to achieve the following in R33 phase of the work: (3) Determine the high-resolution X-ray structure of CXCR4, and (4) Assess the utility of new molecular tools and technologies in stabilizing and crystallizing CXCR4 in complex with gp120. Success of the R21 studies will be achieved by the production of diffracting CXCR4 crystals along with the production of new compound tools that significantly improves receptor sample behavior (e.g. improved stability over time, increased Tm). PUBLIC HEALTH RELEVANCE: Availability of a 3D structure of CXCR4 along with its complexes will lead to developing a deeper understanding of how the HIV virus enters the cell and will help in the design of new therapeutics. CXCR4 is also involved in important physiological process and have been implicated in a number of diseases such as cancer and availability of its structure will likewise have important biomedical implications.