Activation trajectories of integrin α5β1

抽象的。 整合素是介导细胞与其环境之间的许多相互作用的细胞表面受体。 整合素α5β1与其配体纤连蛋白在细胞外基质中的结合在细胞凋亡中起着重要作用。 粘附和分化。在血细胞中，这些相互作用介导生物过程，包括 红细胞生成和单核细胞粘附，并导致病理学，如镰状细胞病，失调 动脉粥样硬化和纤维化。整合素α5β1经历两种不同的构象变化： 在“膝盖”处的延伸和配体结合头盔的开口。这些变化产生了一个整体 在细胞表面上的三种相互转换的整合素构象状态：低亲和力的封闭的（BC）和 延伸闭合（EC）构象和高亲和力延伸开放（EO）构象。这项建议 利用了之前的奖项下的突破性工作，我们在该奖项中测量了自由能和固有能量。 每个整合素α5β1状态的亲和力。使用与用于这些平衡测量相同的Fab工具， 稳定的扩展，关闭，或开放的α5β1构象，我们现在提出三个目标。在目标1中，我们探索 Mn 2+、高Mg 2+和低Ca 2+浓度如何激活整合素。初步结果表明，Mn ~（2+） 和高Mg 2+都增加了EO态的数量，并增加了其对配体的固有亲和力， 这些作用依赖于ADMIDAS金属离子结合位点。为了研究为什么细胞表面α5β1 在BC状态下稳定，我们检验了α和β亚基TM结构域彼此分离的假设 在欧盟和欧盟国家。目的2和3测量动力学以绘制整联蛋白α5β1的活化轨迹， 即在静息状态之间发生的配体结合和构象变化事件的顺序， 当99.8%的未配体整合素α5β1处于BC状态时，最终的功能性配体EO状态（EO·L） 与纤连蛋白结合并通过肌动蛋白施加于整联蛋白的张力稳定的状态 逆行流动并被基质中的纤维连接蛋白抵抗。在目标2中，我们测量了内在配体结合 每个状态的动力学（kon和koff）。我们的初步数据表明，令人惊讶的是，低亲和力的BC和EC 状态比EO状态更快地与配体结合，这通过> 10，000倍的较慢解离速率来补偿 的EO状态。在目标3中，我们使用单分子荧光技术测量整合素构象变化的动力学。 FRET探针测量在构象存在或不存在的情况下的延伸或打开步骤， 特异性Fab和配体。未配体和未配体的BC、EC和EO状态之间的所有转变的动力学 将测定纯化的可溶性胞外域和完整的整合素分子的配体结合的单个整合素分子 使用具有高时间分辨率的TIRF显微术对血细胞上的整合素进行分析。我们希望能找到一种整合素 激活轨迹，其中配体结合到BC+EC状态，然后是配体促进的构象 转化为EO·L状态，随后细胞骨架衔接子（A）结合并稳定EO·L·A状态 通过细胞骨架施加的力和细胞外配体的抵抗。

Abstract. Integrins are cell surface receptors that mediate numerous interactions between cells and their environment. Binding of integrin α5β1 to its ligand fibronectin in the extracellular matrix plays fundamental roles in cellular adhesion and differentiation. In blood cells these interactions mediate biological processes including erythropoiesis and monocyte adhesion and contribute to pathologies such as sickle cell disease, dysregulation of hematopoiesis, atherosclerosis, and fibrosis. Integrin α5β1 undergoes two distinct conformational changes: extension at the ‘knees’ and opening of the ligand-binding headpiece. These changes give rise to an ensemble of three interconverting integrin conformational states on cell surfaces: low-affinity bent-closed (BC) and extended-closed (EC) conformations and a high-affinity extended-open (EO) conformation. This proposal leverages ground-breaking work under the previous award in which we measured free energy and intrinsic affinity of each integrin α5β1 state. Using the same Fab tools as used for these equilibrium measurements to stabilize the extended, closed, or open α5β1 conformations, we now propose three aims. In Aim 1, we explore how Mn2+, high Mg2+, and low Ca2+ concentrations activate integrins. Our preliminary results show that Mn2+ and high Mg2+ both increase the population of the EO state and increase its intrinsic affinity for ligand and that these effects are dependent on the ADMIDAS metal-ion binding site. To examine why cell surface α5β1 is so stable in the BC state, we test the hypothesis that the α and β-subunit TM domains separate from one another in both the EC and EO states. Aims 2 and 3 measure kinetics to map the activation trajectory of integrin α5β1, i.e. the sequence of ligand binding and conformational change events that occur between the resting state, when 99.8% of unliganded integrin α5β1 is in the BC state, and the final, functional liganded EO state (EO•L) state that is bound to fibronectin and is stabilized by tensile force that is applied to the integrin by actin retrograde flow and resisted by fibronectin in the matrix. In Aim 2, we measure the intrinsic ligand-binding kinetics of each state (kon and koff). Our preliminary data indicates, surprisingly, that the low-affinity BC and EC states bind more rapidly to ligand than the EO state, which is compensated by the >10,000-fold slower off-rate of the EO state. In Aim 3, we measure the kinetics of integrin conformational change using single-molecule FRET probes that measure either the extension or opening steps in the presence or absence of conformation- specific Fabs and ligand. Kinetics of all transitions between the BC, EC, and EO states for unliganded and ligand-bound single integrin molecules will be determined for both purified, soluble ectodomain and intact integrins on blood cells using TIRF microscopy with high temporal resolution. We expect to show an integrin activation trajectory in which ligand binds to the BC+EC states, followed by ligand-facilitated conformational conversion to the EO•L state, followed by cytoskeletal adaptor (A) binding and stabilization of the EO•L•A state by force applied by the cytoskeleton and resisted by extracellular ligand.