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，使粘附蛋白在其力激活，扩展状态构象中的晶体结构研究成为可能。对粘附复合物生理（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物的调控的理解，并使更有效的药物治疗和预防疾病。具体而言，申请人将研究血管性血液病因子a1结构域（VWF-A1）与血小板糖蛋白GPIba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。我们假设在VWF-A1和GPIba之间存在一种力激活的扩展态复合体。如果没有扩展态结构的先验知识，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对蛋白质晶体施加力。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哪些突变来稳定扩展状态，申请人将使用随机和聚焦诱变结合酵母表面显示来筛选高亲和力结合物（即激活）。然后，申请人将(2)结合VWF-A1和GPIba的突变来发现潜在的协同效应。接下来，申请人将(3)利用流式细胞术和表面等离子体共振表征假定的扩展状态突变对的结合亲和力和动力学。(4)突变对黏附动力学和力学稳定性的影响将在平行板流室研究中进行。最后，申请人将(5)结晶扩展态复合物以进行结构阐明并与野生型和血管性血友病突变体进行比较。

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.