Structure Analysis of Integrin Activation

描述(申请人提供)：心血管疾病是美国人死亡的主要原因，主要是由于动脉粥样硬化斑块破裂和随后的冠状动脉内血栓形成引起的心肌梗死。刺激血栓形成的一个关键事件是血小板聚集，它是由典型的整合素AIIB介导的。整合素是一个异二聚体跨膜受体家族，调节细胞黏附，如血小板聚集，以及其他重要的生物过程，如细胞迁移、分化、增殖和程序性细胞死亡。整合素通过调节细胞外黏附分子和细胞内环境之间的动态联系来实现这些不同的功能。整合素的功能受跨膜信号的调节，这可能是结合细胞外配体(“由外而内”的信号传递)以及分子与细胞质结构域结合(“由内而外”的信号传递)的结果。我们建议使用电子冷冻显微镜、图像重建和分子建模来研究人血小板整合素AIIB的结构。电子冷冻显微镜(Cryo-EM)是一种强大的技术，通过它可以在自然的生理状态下检查膜蛋白等大分子复合体。我们已经完成了低亲和力、非活性构象的全长人血小板整合素AIIB？3的3D结构。然后将AV3胞外区的X射线结构对接到低分辨率冷冻EM图中。这种结合的方法使我们能够为与整合素激活相关的结构重排提出一个模型。这个项目的总体目标是测试这个整合素激活模型，我们的实验按照以下具体目标组织：目标1：将继续进行图像分析，以提高低亲和力状态下亚投行3的三维重建的分辨率；目标2：通过分析具有结合配体的亚投行3，得到高亲和力状态的3D地图；目标3：将使用抗体标记来定位高亲和力状态和低亲和力状态中的特定位置；目的：结合冷冻-EM图谱、位点特异性抗体标记数据和单个整合素结构域的同源模型，得到高亲和力状态和低亲和力状态的分子模型。这些研究揭示的结构细节将为整合素激活的分子基础提供洞察，这与新治疗剂的设计相关。

DESCRIPTION (provided by applicant): Cardiovascular disease is the major cause of mortality in the United States, primarily due to myocardial infarction resulting from rupture of an atherosclerotic plaque and subsequent thrombosis within a coronary artery. A key event that stimulates thrombus formation is platelet aggregation, which is mediated by the prototypical integrin aIIb¿3. Integrins are a family of heterodimeric transmembrane receptors which modulate cell adhesion, such as platelet aggregation, as well as other important biological processes such as cell migration, differentiation, proliferation and programmed cell death. Integrins accomplish these diverse functions by mediating dynamic linkages between extracellular adhesion molecules and the intracellular environment. Integrin functions are regulated by transmembrane signaling, which can occur as a consequence of binding extracellular ligands ("outside-in" signaling), as well as the binding of molecules to the cytoplasmic domains ("inside-out" signaling). We propose to use electron cryo-microscopy, image reconstruction and molecular modeling to examine the structure of the human platelet integrin aIIb¿3. Electron cryo-microscopy (cryo-EM) is a powerful technique by which macromolecular complexes such as membrane proteins can be examined in a native, physiological state. We have completed a 3D structure of the full-length, human platelet integrin aIIb¿3 in the low-affinity, inactive conformation. The X-ray structure of the aV¿3 ectodomain was then docked into the low resolution cryo-EM map. This combined approach allowed us to propose a model for the structural rearrangements associated with integrin activation. The overall goal of this project is to test this model for integrin activation, and our experiments are organized according to the following specific aims: Aim 1: Image analysis will be continued in order to improve the resolution of the 3D reconstruction of aIIb¿3 in the low-affinity state; Aim 2: A 3D map of the high-affinity state will be derived by analysis of aIIb¿3 with bound ligands; Aim 3: Antibody labeling will be used to localize specific sites within the high- and low-affinity states; Aim 4: Molecular models of the high- and low- affinity states will be derived by combining the cryo-EM maps, site-specific antibody labeling data, and homology models for individual integrin domains. The structural details revealed by these studies will provide insight into the molecular basis of integrin activation, relevant for the design of new therapeutic agents.