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: 6271781
• 负责人: ELIZABETH A. KOMIVES
• 金额: $ 5.46万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6271781
• 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 血栓形成蛋白（TM）与凝血酶可能导致新颖的发展 治疗心脏病的治疗剂。 TM的结合 凝血酶会抑制凝血酶的凝凝术活性 （纤维蛋白原的切割）和抗凝剂的选择性增强 凝血酶的活性（蛋白C的激活）。先前的研究已有 表明包含表皮表皮的三个域的TM区域 生长因子（EGF域）是必要的，足以与 凝血酶并改变凝血酶的催化活性。我们的实验会 确定TM EGF域相互作用的分子细节 与凝血酶和蛋白质的同时激活。 TM的第四，第五和第六EGF域 在蛋白C的凝血酶裂解的结合和激活中 在大肠杆菌中表达，大量纯化域将是 获得。 我们将研究每个域和对的功能 通过测定抑制TM-凝血酶的能力来分析域的域 络合或激活凝血酶以催化蛋白质的裂解 C 功能上重要域的解决方案结构将是 由二维NMR方法确定。然后我们将执行 测量转移的核透射效应对质子的效应 凝血酶确定TM相互作用的分子细节 EGF域具有凝血酶。我们还将尝试共结合域 使用凝血酶获得有关蛋白质蛋白的结构信息 互动。从序列相似性和类比到 表皮生长因子与其受体的相互作用表明这两种 beta转向区域参与了相互作用。这个假设将是 使用定向诱变进行探测。