Enabling Structural Studies of Force Activated Adhesion Complexes

实现力激活粘附复合物的结构研究

• 批准号: 8193470
• 负责人: Mark Alan Blenner
• 金额: $ 3.37万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8193470
• 关键词: Adhesions; Affinity; Binding; Complex; Coupled; Disease; Flow Cytometry; Hemorrhage; Hemostatic Agents; Hemostatic function; Kinetics; Knowledge; Lead; Ligands; Mechanics; Methods; Molecular; Molecular Conformation; Mutagenesis; Mutation; Pharmaceutical Preparations; Physiological; Platelet Glycoproteins; Play; Process; Proteins; Regulation; Research; Role; Structure; Surface; Surface Plasmon Resonance; Testing; Yeasts; effective therapy; hemodynamics; mutant; prevent; public health relevance; von Willebrand Disease; von Willebrand Factor

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.

描述（由申请人提供）：所提议研究的广泛、长期目标是开发一种方法，能够研究粘附蛋白在力激活、延伸态构象下的晶体结构。对粘附复合物生理（或病理）状态的原子水平了解应该增加我们对力激活蛋白-配体复合物调节的理解，并使更有效的药物能够治疗和预防疾病。具体来说，申请人将研究血管性血友病因子的A1结构域（VWF-A1）和血小板糖蛋白GPIb a结构域（GPIba）之间的相互作用。这种相互作用是由血液动力学流动中的剪切力激活的，对于止血很重要。我们假设 VWF-A1 和 GPIba 之间存在力激活的扩展状态复合体。如果没有扩展态结构的先验知识，目前不可能获得这些复合物的晶体结构，因为没有生理上有意义的方法向蛋白质晶体施加力。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，目的是在没有外力的情况下获得这些难以捉摸的晶体结构。该研究的具体目标是 (1) 寻找对 VWF-A1 和 GPIba 具有增强亲和力的突变体。由于需要进行哪些突变来稳定延伸状态并不明显，因此申请人将使用随机和集中诱变与酵母表面展示相结合来筛选高亲和力结合物（即，活化的）。然后，申请人将 (2) 组合 VWF-A1 和 GPIba 的突变以发现潜在的协同效应。接下来，申请人将(3)使用流式细胞术和表面等离子体共振来表征推定的延伸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)结晶扩展态复合物以进行结构阐明并与野生型和冯维勒布兰德病突变体进行比较。 公共健康相关性：血管性血友病因子在身体止血能力中发挥着至关重要的作用。为了更好地了解这一过程以及伤口无法止血的疾病，我们寻求分子水平上的血管性血友病因子如何发挥其止血作用的知识。这种理解可能会带来更有效的出血性疾病治疗方法。