STRUCTURES OF CATALYTIC DOMAINS OF BLOOD PROTEINS

血液蛋白催化域的结构

• 批准号: 3361804
• 负责人: ALEXANDER TULINSKY
• 金额: $ 13.1万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3361804
• 关键词: X ray crystallography; conformation; crystallization; enzyme mechanism; enzyme structure; fibrinogen; hirudins; peptidases; protein folding; protein structure; prothrombin; thrombin

The immediate objective of this work is to solve and refine the structure of human PPACK-alpha-thrombin. The structure of this prototype of serine protease catalytic domains will reveal the manner in which the highly selective fibrinogen-thrombin interaction is accomplished. Moreover, since many of the enzyme domains of blood coagulation/fibrinolysis are highly homologous, the human alpha-thrombin structure will be used to model closely homologous regions of these other proteases as well as model differences between species. The structure of alpha-thrombin will then be used in molecular replacement application to solve the structures of the hirudin complexes of alpha- and PPACK-alpha-thrombin. These studies are designed to localize the binding sites of the anti-thrombotic hirudin and the thrombin molecules to delineate the three dimensional and other factors responsible for the incredibly singular binding affinity between these molecules (femto and pico molar, respectively). Similar methods will be employed to determine the structures of prothrombin 1 and single chain prothrombin 2. In the case of the former, rotation-translation searches will also include the three disulfide, triple loop of the kringle 2- catalytic domain interactions of prothrombin; a comparison of the pre- thrombin 2 and thrombin structures will reveal the conformational changes occurring upon activation cleavage of prothrombin by Factor Xa. A longer range thrust of the work will center upon the structure determination of intact cassette domain molecules such as the thrombolytics tissue plasminogen activator (TPA) and prourokinase. The scope of the effort will be broadened and enhanced by also attempting the crystallization of third generation, deglycosylated exon shuffled constructs of the TPA molecule. The number of possibilities of interchanging and/or deleting finger, growth factor, kringle and catalytic domains is attractively large thus favorably increasing the probability of uncovering crystallizable constructs.

这项工作的直接目的是解决和完善结构 人类ppack-alpha-thrombin。 这个丝氨酸原型的结构 蛋白酶催化域将揭示高度的方式 选择性纤维蛋白原 - 凝血酶蛋白相互作用已完成。 而且， 由于许多血液凝血/纤维蛋白溶解的酶结构域都是 高度同源的人α-凝血酶结构将用于 模型与这些其他蛋白酶的紧密同源区域以及 物种之间的模型差异。 然后，α-凝血酶的结构将用于分子 替换应用以解决Hirudin复合物的结构 α-和ppack-alpha-thrombin。 这些研究被设计为 定位抗凝血蛋白的结合位点和 凝血酶分子描绘三维和其他因素 负责这些之间令人难以置信的奇异绑定亲和力 分子（分别为femto和pico molar）。 类似的方法将是 用于确定凝血酶原1和单链的结构 凝血酶原2。对于前者，旋转翻译搜索 还将包括Kringle 2-的三个二硫键三重环 凝血酶原的催化结构域相互作用；比较 凝血酶2和凝血酶结构将揭示构象变化 发生在因子Xa的激活后凝血酶激活后发生。 工作的更长范围将以结构为中心 确定完整的盒式结构域分子，例如 溶栓组织纤溶酶原活化剂（TPA）和prourokinase。 这 努力的范围将通过尝试来扩大和增强 第三代，脱糖外显子的结晶 TPA分子的构建体。 可能性的数量 互换和/或删除手指，生长因子，kringle和 催化结构域具有吸引力，因此有利地增加 发现可结晶的构建体的概率。