Atomic Resolution Characterization of Kindlin-2 Binding to Phosphatidylinositol Phosphatases in Lipid Bilayers by Solid-State NMR

项目摘要： Kindlin-2（K2）是调节整合素的外周膜蛋白，整合素是介导细胞凋亡的关键蛋白。 人体内的粘连。该项目的目标是增加我们对K2和脂质的结构理解 它结合的膜，提供关键的见解K2的调节功能。K2的相互作用 与细胞膜的关系很难研究，因为膜是高度动态的，缺乏长程有序性。 因此，我们研究膜和膜蛋白的能力受到限制，尽管它们具有潜力， 在广泛的系统中发挥影响力。在这个建议中，我们将集中在信号脂质磷脂酰肌醇 磷酸盐（PIP）控制K2的活性。这种脂质家族对于人类健康是重要的，因为其作用 PIP作为许多细胞过程的调节剂，包括营养感测和生长。监管不当 PIP依赖性途径与许多罕见和常见疾病有关，包括癌症和糖尿病。 PIP激活的机制细节和蛋白质结合伴侣（如K2）的调节对于 了解和防治这些疾病。固体核磁共振是一种独特的原子分辨工具 表征脂质的结构和动力学，并确定控制脂质-蛋白质的因素 交互.在这个提议的五年时间轴里，我计划开发核磁共振实验， 以高灵敏度和特异性研究K2-脂质界面。本研究的重点将是保守的 K2的PIP结合结构域：泛素样F0结构域和普列克底物蛋白同源（PH）结构域。的存在 PIP中的天然NMR活性和化学上不同的磷酸基团将用作直接 探测结合域-脂质界面。脂质头部基团与 蛋白质将是我们研究PIP识别的结构基础和机制的基础。 K2的激活。除了研究蛋白质-脂质界面，我们还试图了解膜是如何 结合影响K2与伴侣蛋白及其自身的相互作用。我们的实验将充分利用 NMR硬件的最新改进，特别是非常快速的魔角旋转。我和我的团队 在开发新的实验来分配化学位移和解决蛋白质的结构方面经验丰富 复合物、膜蛋白和蛋白原纤维。这为我们提供了解决这些问题所需的工具。 脂质-蛋白质和蛋白质-蛋白质界面的结构方面。我们将采用综合方法， 最先进的计算技术将指导我们的实验工作全原子分子动力学将是 用于提供可以用NMR测试的假设，并提供 实验数据这项工作对于帮助设计靶向K2的治疗和诊断至关重要， 其他PIP绑定系统。该项目的结果将提高我们描述K2-PIP相互作用的能力， 了解致病突变对整合素调节的影响，并提出解决方案， 人类健康面临的一些最大挑战。

Project Summary: Kindlin-2 (K2) is a peripheral membrane protein which regulates integrin, a key protein that mediates cellular adhesion in humans. The goal of this project is to increase our structural understanding of K2 and the lipid membranes to which it binds, to provide key insights into the regulatory function of K2. The interactions of K2 with cellular membranes are difficult to study because membranes are highly dynamic and lack long-range order. As a result, our ability to study membranes and membrane proteins have been limited despite their potential to be impactful in a wide array of systems. In this proposal we will focus on the signaling lipids phosphatidylinositol phosphates (PIPs) which control the activity of K2. This lipid family is important for human health due to the role of PIPs as regulators of numerous cell processes including nutrient sensing and growth. The misregulation of PIP-dependent pathways is implicated in numerous rare and common diseases, including cancer and diabetes. Mechanistic details of PIP activation and regulation of protein binding partners, like K2, are essential to understanding and combating these diseases. Solid-state NMR is a unique tool for the atomic resolution characterization of the structure and dynamics of lipids and determining the factors that govern lipid-protein interactions. Over the five-year timeline of this proposal I plan to develop NMR experiments for directly investigating K2-lipid interfaces with high sensitivity and specificity. The focus of this study will be the conserved PIP binding domains of K2: a ubiquitin-like F0 domain and a pleckstrin homology (PH) domain. The presence of naturally NMR-active and chemically distinct phosphate groups in the PIPs will serve as a handle to directly probe the binding domain-lipid interface. Characterization of specific interactions between lipid head groups and proteins will be the foundation upon which we examine the structural basis of PIP recognition and the mechanism of K2 activation by PIPs. Beyond studies of the protein-lipid interface, we seek to understand how membrane binding affects the interactions of K2 with partner proteins and itself. Our experiments will take advantage of all the latest improvements in NMR hardware, particularly very-fast magic angle spinning. My group and I are highly experienced in developing novel experiments to assign the chemical shifts and solve structures of protein complexes, membrane proteins, and protein fibrils. This provides us with the tools that are required to tackle the structural aspects of lipid-protein and protein-protein interfaces. We will use an integrative approach in which state-of-the-art computational techniques will guide our experimental work. All-atom molecular dynamics will be used to provide hypotheses that may be tested with NMR and to provide atomic resolution interpretations of experimental data. This work is vital for assisting in the design of therapeutics and diagnostics that target K2 and other PIP binding systems. The results of this project will improve our ability to describe K2-PIP interactions, understand the effects of disease-causing mutations on the regulation of integrins, and propose solutions to some of the largest challenges in human health.