MECHANISTIC INSIGHTS INTO THE ?INSIDE-OUT? SIGNALING OF INTEGRINS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 我们研究活动的总体目标是实现对膜蛋白复杂系统中分子识别触发的信号转导过程的详细机制理解。我们实验室资助的一个研究项目集中在血小板整合素？IIB？3，这是一种膜异二聚体蛋白，通过结合粘附性血浆蛋白纤维蛋白原和von Willebrand因子来介导血小板聚集。这种蛋白在血管损伤部位止血方面起着重要作用，但也在血栓形成中起着重要作用，血栓形成可能导致心脏病发作和中风。与其他整合素一样，βIIB？3整合素可以通过其短的细胞质尾巴与细胞内蛋白质如talin的相互作用(由内向外信号传递)、或其胞外结构域与细胞外基质成分或可溶性配体的相互作用(由外向内信号传递)引起的长程变构变化而产生双向信号。整合素跨膜(TM)螺旋之间的相互作用？然后呢？亚基被认为在整合素的激活和聚集中发挥重要作用。值得注意的是，TM螺旋被认为在整合素静止状态下相互作用，但在Talin诱导的整合素激活和细胞黏附时分离。然而，整合素TM螺旋之间的特异性和亲和力一直很难证明，并与激活过程有关。最近从溶液核磁共振和X射线散射实验中得到的模型阐明了整合素TM相互作用的一些细节，但也提出了关于整合素信号处理的原子水平机制的问题。我们建议研究整合素βIIB和β3亚基在Talin存在或不存在的情况下TM区相互作用的动力学细节，最终目的是了解talin导致TM二聚化破坏的分子机制。从计算的角度来看，理解整合素TM二聚化的结构和热力学特征不是一个微不足道的问题。这在很大程度上是由于这一生物过程的随机性，以及随后需要对显式脂-水环境中蛋白质复合体的完全原子化表示进行多个长时间尺度的分子动力学(MD)模拟。Anton具有独特的定位，能够提供有关整合素TM区域二聚化的详细结构和动力学信息。因此，我们建议在Anton上进行多微秒尺度的分子动力学模拟，其结果有望为研究整合素？IIB？3的“内向外”信号提供突破性的见解。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The overall goal of our research activities is to achieve a detailed mechanistic understanding of signal transduction processes triggered by molecular recognition in membrane protein complex systems. One of the funded research programs in our lab focuses on platelet integrin ?IIb?3, a membrane heterodimeric protein that mediates platelet aggregation by binding the adhesive plasma proteins fibrinogen and von Willebrand factor. This protein plays an essential role in initiating arrest of bleeding at sites of vascular injury, but also in thrombus formation, which may lead to heart attacks and stroke. Like other integrins, ?IIb?3 integrin can signal bidirectionally through long-range allosteric changes that are induced by the interactions of their short cytoplasmic tails with intracellular proteins such as talin (inside-out signaling), or by interactions of their extracellular domains with extracellular matrix components or soluble ligands (outside-in signaling). Interactions between the transmembrane (TM) helices of integrin ? and ? subunits have been suggested to play an important role in integrin activation and clustering. Notably, the TM helices are believed to interact in the integrin resting state, but to separate upon talin-induced integrin activation and cellular adhesion. However, the specificity and affinity between integrin TM helices have been difficult to demonstrate, and to relate to the activation process. Recent models derived from solution NMR and X-ray scattering experiments have clarified some details of the integrin TM interaction, but also raised questions about the atomic-level mechanism of the integrin signal processing. We propose to study the dynamical details of the interaction of the TM regions of integrin ?IIb and ?3 subunits in the presence or absence of talin, with the ultimate goal of understanding the molecular mechanism by which talin causes TM dimerization disruption. From a computational perspective, understanding the structural and thermodynamic features of integrin TM dimerization is not a trivial problem. To a large extent, this is due to the stochastic nature of this biological process, and the consequent need to perform multiple long-timescale molecular dynamics (MD) simulations of fully atomistic representations of protein complexes in an explicit lipid-water environment. Anton is uniquely positioned to provide detailed structural and dynamical information about the dimerization of integrin TM regions. Thus, we propose to carry out on Anton multiple microsecond-scale MD simulations, whose results are expected to provide breakthrough insights into the "inside-out" signaling of ?IIb?3 integrins.