Stoop A A, Eldering E, Dafforn T R, Read R J, Pannekoek H
Department of Biochemistry Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
J Mol Biol. 2001 Jan 26;305(4):773-83. doi: 10.1006/jmbi.2000.4356.
Plasminogen activator inhibitor type 1 (PAI-1) is a member of the serine protease inhibitor (serpin) superfamily. Its highly mobile reactive-center loop (RCL) is thought to account for both the rapid inhibition of tissue-type plasminogen activator (t-PA), and the rapid and spontaneous transition of the unstable, active form of PAI-1 into a stable, inactive (latent) conformation (t(1/2) at 37 degrees C, 2.2 hours). We determined the amino acid residues responsible for the inherent instability of PAI-1, to assess whether these properties are independent and, consequently, whether the structural basis for inhibition and latency transition is different. For that purpose, a hypermutated PAI-1 library that is displayed on phage was pre-incubated for increasing periods (20 to 72 hours) at 37 degrees C, prior to a stringent selection for rapid t-PA binding. Accordingly, four rounds of phage-display selection resulted in the isolation of a stable PAI-1 variant (st-44: t(1/2) 450 hours) with 11 amino acid mutations. Backcrossing by DNA shuffling of this stable mutant with wt PAI-1 was performed to eliminate non-contributing mutations. It was shown that the combination of mutations at positions 50, 56, 61, 70, 94, 150, 222, 223, 264 and 331 increases the half-life of PAI-1 245-fold. Furthermore, within the limits of detection the stable mutants isolated are functionally indistinguishable from wild-type PAI-1 with respect to the rate of inhibition of t-PA, cleavage by t-PA, and binding to vitronectin. These stabilizing mutations constitute largely reversions to the stable "serpin consensus sequence" and are located in areas implicated in PAI-1 stability (e.g. the vitronectin-binding domain and the proximal hinge). Collectively, our data provide evidence that the structural requirements for PAI-1 loop insertion during latency transition and target proteinase inhibition can be separated.
纤溶酶原激活物抑制剂1型（PAI-1）是丝氨酸蛋白酶抑制剂（serpin）超家族的成员。其高度可移动的反应中心环（RCL）被认为是组织型纤溶酶原激活物（t-PA）快速抑制以及PAI-1不稳定的活性形式向稳定的无活性（潜伏）构象快速自发转变（37℃时的半衰期t(1/2)为2.2小时）的原因。我们确定了导致PAI-1固有不稳定性的氨基酸残基，以评估这些特性是否独立，进而评估抑制和潜伏转变的结构基础是否不同。为此，在严格筛选快速t-PA结合之前，将展示在噬菌体上的超突变PAI-1文库在37℃下预孵育不同时间（20至72小时）。相应地，四轮噬菌体展示筛选导致分离出一种具有11个氨基酸突变的稳定PAI-1变体（st-44：t(1/2)为450小时）。通过该稳定突变体与野生型PAI-1的DNA改组进行回交以消除无作用的突变。结果表明，50、56、61、70、94、150、222、223、264和331位的突变组合使PAI-1的半衰期增加了245倍。此外，在所检测的范围内，分离出的稳定突变体在t-PA抑制率、t-PA切割以及与玻连蛋白结合方面在功能上与野生型PAI-1没有区别。这些稳定突变在很大程度上恢复为稳定的“serpin共有序列”，并且位于与PAI-1稳定性相关的区域（例如玻连蛋白结合结构域和近端铰链区）。总体而言，我们的数据提供了证据，表明PAI-1在潜伏转变期间环插入和靶蛋白酶抑制的结构要求是可以分开的。
