BIOPHYSICAL STUDIES OF THE THROMBOMODULIN EGF DOMAINS

血栓调节蛋白 EGF 结构域的生物物理学研究

• 批准号: 6240513
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 1.67万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240513
• 关键词: X ray crystallography; antithrombins; binding proteins; chemical binding; enzyme induction /repression; epidermal growth factor; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein C; protein purification; protein structure function; site directed mutagenesis; thrombomodulin

Knowledge of the details of the protein-protein interaction of thrombomodulin (TM) with thrombin may lead to the development of novel therapeutic agents for the treatment of heart disease. The binding of TM to thrombin causes an inhibition of the procoagulant activity of thrombin (cleavage of fibrinogen) and a selective enhancement of the anticoagulant activity of thrombin (activation of protein C). Previous studies have shown that a region of TM containing three domains that resemble epidermal growth factor (EGF domains) are necessary and sufficient to bind to thrombin and to alter thrombin's catalytic activity. Our experiments will determine the molecular details of the interaction of the TM EGF domains with thrombin, and of the concomitant activation of protein C. The fourth, fifth, and sixth EGF domains from TM that have been implicated in the binding and activation of thrombin cleavage of protein C will be expressed in E. coli, and large amounts of the purified domains will be obtained. We will study the function of each of these domains and pairs of domains by assaying their ability to either inhibit the TM-thrombin complexation, or to activate thrombin to catalyze the cleavage of protein C. The solution structures of the functionally important domains will be determined by two-dimensional NMR methods. We will then carry out measurements of transferred nuclear Overhauser effects to protons on thrombin to determine the molecular details of the interaction of the TM EGF domains with thrombin. We will also try to co-crystallize the domains with thrombin to gain structural information about the protein-protein interactions. Information from sequence similarity and analogy to the interaction of epidermal growth factor with its receptor suggests the two beta-turn regions are involved in the interaction. This hypothesis will be probed using site-directed mutagenesis.

了解蛋白质-蛋白质相互作用的细节 血栓调节蛋白（TM）与凝血酶可能会导致新型药物的开发 用于治疗心脏病的治疗剂。 TM 的绑定 凝血酶导致凝血酶促凝血活性的抑制 （纤维蛋白原的裂解）和抗凝剂的选择性增强 凝血酶的活性（蛋白 C 的激活）。之前的研究有 结果表明，TM 的一个区域包含三个类似于表皮的结构域 生长因子（EGF 结构域）对于结合是必要且充分的 凝血酶并改变凝血酶的催化活性。我们的实验将 确定 TM EGF 结构域相互作用的分子细节 与凝血酶的作用，以及蛋白 C 的伴随激活。 TM 中涉及的第四、第五和第六个 EGF 结构域 在蛋白质 C 的凝血酶裂解的结合和激活中 在大肠杆菌中表达，大量的纯化结构域将被 获得。 我们将研究每个域和对的功能 通过测定域抑制 TM 凝血酶的能力 络合，或激活凝血酶以催化蛋白质裂解 C. 功能重要领域的解决方案结构将是 通过二维NMR方法测定。然后我们将进行 测量质子的转移核奥沃豪瑟效应 凝血酶以确定 TM 相互作用的分子细节 EGF 结构域与凝血酶。我们还将尝试共结晶域 与凝血酶一起获得有关蛋白质-蛋白质的结构信息 互动。来自序列相似性和类比的信息 表皮生长因子与其受体的相互作用表明两者 β-转角区域参与相互作用。这个假设将是 使用定点诱变进行探测。