Enabling Structural Studies of Force Activated Adhesion Complexes

描述(由申请人提供)：拟议研究的广泛的、长期的目标是开发一种方法来研究粘着蛋白在其力激活的扩展状态构象中的晶体结构。对黏附复合体的生理(或病理)状态的原子水平的了解将增加我们对力激活的蛋白质-配体复合体的调节的理解，并使更有效的药物能够治疗和预防疾病。具体地说，申请人将研究Von Willebrand因子(VWF-A1)的A1结构域与血小板膜糖蛋白GPIb a结构域(GPIba)之间的相互作用。这种相互作用是由血流动力学中的剪切力激活的，对止血很重要。我们假设VWF-A1和GPIba之间存在一个力激活的扩展态复合体。如果没有扩展态结构的先验知识，目前获取这些复合体的晶体结构是不可能的，因为没有对蛋白质晶体施加力的生理意义的方法。因此，我们建议开发一种方法来识别稳定扩展态复合体的突变，目的是在无力的情况下获得这些难以捉摸的晶体结构。本研究的具体目的是(1)寻找与VWF-A1和GPIba具有更高亲和力的突变体。由于尚不清楚需要进行哪些突变来稳定扩展状态，申请人将使用随机和集中突变的组合，结合酵母表面展示来筛选高亲和力结合物(即，激活的)。申请者随后将(2)将VWF-A1和GPIba的突变结合起来，以揭示潜在的协同效应。接下来，申请者将(3)使用流式细胞术和表面等离子体共振来表征假定的扩展状态突变对的结合亲和力和动力学。(4)突变对粘着动力学和机械稳定性的影响将在平行板流室研究中进行。最后，申请者将(5)结晶扩展状态的复合体，用于结构说明和与野生型和Von Willebrand病突变体的比较。 公共卫生相关性：von Willebrand因子在人体止血的能力中发挥着关键作用。为了更好地了解这一过程以及伤口无法止血的疾病，我们寻求关于Von Willebrand因子如何发挥止血作用的分子水平的知识。这一认识可能会导致更有效的治疗出血性疾病。

DESCRIPTION (provided by applicant): The broad, long-term objective of the proposed research is to develop a method to enable crystal structure studies of adhesion proteins in their force activated, extended-state conformation. Atomic level knowledge of the physiological (or pathological) state of adhesion complexes should increase our understanding of the regulation of force activated protein-ligand complexes and enable more effective drugs to treat and prevent disease. Specifically, the applicant will study the interaction between the A1-domain of Von Willebrand Factor (VWF-A1) and platelet glycoprotein GPIb a-domain (GPIba). This interaction is activated by shear forces in hemodynamic flow and is important for hemostasis. We hypothesize the existence of a force-activated, extended-state complex between VWF-A1 and GPIba. Without a priori knowledge of the extended-state structure, obtaining crystal structures of these complexes is currently impossible since there is no physiologically meaningful way to apply force to a protein crystal. Therefore, we propose to develop a method to identify mutations that stabilize the extended-state complex with the purpose of obtaining these elusive crystal structures in the absence of force. The specific aims of the research are to (1) Find mutants with enhanced affinity for both VWF-A1 and GPIba. Since it is not obvious what mutations need to be made to stabilize the extended-state, the applicant will use a combination of random and focused mutagenesis coupled with yeast surface display to screen for high affinity binders (i.e., activated). The applicant will then (2) Combine mutations in VWF-A1 and GPIba to uncover potential synergistic effects. Next, the applicant will (3) Characterize the binding affinity and kinetics of the putative extended-state mutant pairs using flow cytometry and surface plasmon resonance. (4) The mutations' effects on adhesion dynamics and mechanical stability will be studied in parallel plate flow chamber studies. Last, the applicant will (5) Crystallize the extended-state complex for structural elucidation and comparison to wild-type and Von Willebrand Disease mutants. PUBLIC HEALTH RELEVANCE: Von Willebrand Factor plays a crucial role in the body's ability to cease bleeding from wounds. In order to better understand this process, and diseases where wounds cannot cease bleeding, we seek molecular level knowledge of how Von Willebrand Factor performs its hemostatic role. This understanding may lead to more effective treatments for bleeding disorders.