Enabling Structural Studies of Force Activated Adhesion Complexes

描述（由申请人提供）：拟议研究的广泛、长期目标是开发一种方法，以使粘附蛋白在其力激活、扩展状态构象下的晶体结构研究成为可能。粘附复合物的生理（或病理）状态的原子水平的知识应该增加我们对力激活蛋白质-配体复合物的调节的理解，并使更有效的药物能够治疗和预防疾病。具体而言，申请方将研究血管性血友病因子A1结构域（VWF-A1）与血小板糖蛋白GPIb a结构域（GPIb a）之间的相互作用。这种相互作用由血流动力学流动中的剪切力激活，对止血很重要。我们假设存在一个力激活的，扩展状态复杂的VWF-A1和GPIBA之间。如果没有延伸态结构的先验知识，获得这些复合物的晶体结构目前是不可能的，因为没有生理学上有意义的方法来施加力到蛋白质晶体。因此，我们建议开发一种方法来识别稳定扩展态复合物的突变，目的是在没有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）寻找对VWF-A1和GPIba都具有增强亲和力的突变体。由于不清楚需要进行什么样的突变来稳定延伸状态，申请人将使用随机和聚焦诱变的组合与酵母表面展示结合来筛选高亲和力结合剂（即，激活）。然后申请人将（2）联合收割机组合VWF-A1和GPIba中的突变，以揭示潜在的协同效应。接下来，申请人将（3）使用流式细胞术和表面等离子体共振表征推定的延伸状态突变体对的结合亲和力和动力学。(4)将在平行板流动室研究中研究突变对粘附动力学和机械稳定性的影响。最后，申请人将（5）结晶扩展态复合物，用于结构解析并与野生型和冯维勒布兰德病突变体进行比较。 公共卫生相关性：血管性血友病因子在身体停止伤口出血的能力中起着至关重要的作用。为了更好地了解这一过程，以及伤口无法止血的疾病，我们寻求冯维勒布兰德因子如何发挥其止血作用的分子水平知识。这种理解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.