STRUCTURES OF CATALYTIC DOMAINS OF BLOOD PROTEINS

这项工作的直接目标是解决和完善结构 人类PPACK-α-凝血酶。丝氨酸的这个原型的结构 蛋白水解酶催化结构域将揭示高度 纤维蛋白原-凝血酶的选择性相互作用已经完成。此外， 由于凝血/纤溶的许多酶结构域是 高度同源性，人类的α-凝血酶结构将被用于 对这些其他蛋白水解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 取代法应用于水飞蓟素络合物结构的求解 α-和PPACK-α-凝血酶。这些研究旨在 定位抗血栓水飞蓟素的结合部位 凝血酶分子勾画三维等因素 负责它们之间令人难以置信的单一绑定亲和力 分子(分别为毫微微和微微摩尔)。类似的方法将是 用于测定凝血酶原1和单链的结构 凝血酶原2。在前者的情况下，旋转平移搜索 还将包括Kringle 2的三个二硫键、三重环- 凝血酶原的催化域相互作用； 凝血酶2和凝血酶结构将揭示构象变化 在凝血酶原被凝血因子Xa激活裂解时发生。 这项工作的更大推力将集中在结构上 完整盒结构域分子的测定，如 溶栓剂、组织型纤溶酶原激活剂(TPA)和尿激活酶原。这个 这项工作的范围将扩大和加强，方法是还尝试 第三代脱糖基化外显子的结晶被改组 TPA分子的结构。的可能性的数量 互换和/或删除手指、生长因子、kringle和 催化结构域非常大，因此有利于增加 发现可结晶结构的可能性。

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.