Formation of a noncovalent serpin-proteinase complex involves no conformational change in the serpin. Use of 1H-15N HSQC NMR as a sensitive nonperturbing monitor of conformation.
Formation of a noncovalent serpin-proteinase complex involves no conformational change in the serpin. Use of 1H-15N HSQC NMR as a sensitive nonperturbing monitor of conformation.
复制标题
DOI：
10.1021/bi001152
复制
发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
F. Peterson;N. Gordon;P. Gettins
中科院分区：
文献类型：
--
作者：
F. Peterson;N. Gordon;P. Gettins
对丝氨酸蛋白酶抑制剂抑制机制的性质和范围的结构理解受到迄今为止不能使任何丝氨酸蛋白酶抑制剂-蛋白酶复合物结晶的限制。我们在这里描述的应用[（1）H-（15）N]-HSQC NMR均匀和残基选择性（15）N-标记的丝氨酸蛋白酶抑制剂α（1）-蛋白酶抑制剂（匹兹堡变种稳定突变）提供一个非扰动和精致的敏感手段探测构象的丝氨酸蛋白酶抑制剂单独和非共价复合物与无活性，丝氨酸195-修饰，牛胰蛋白酶。后者应该是一个很好的模型，为可逆的丝氨酸蛋白酶抑制剂-蛋白酶复合物的几个例子，并为最初的米氏样复合物形成途中不可逆的共价抑制。裂解的反应中心环，随后插入β-片层A，造成显着扰动的大部分NMR交叉峰。这对于均匀标记和丙氨酸特异性标记的样品都是正确的。均匀或亮氨酸或丙氨酸特异性标记的α（1）-蛋白酶抑制剂与未标记的非活性胰蛋白酶的非共价复合物的光谱几乎没有检测到的交叉峰的化学位移变化，但一些一般的线宽增加。残基特异性分配的四个丙氨酸在反应中心环，在P12，P11，P9和P4，允许特定的检查反应中心环的行为。所有四个丙氨酸表现出更高的流动性比身体的丝氨酸蛋白酶抑制剂，一个灵活的反应中心环，保持灵活，即使在非共价复合物与蛋白酶一致。三个丙氨酸附近的铰链点插入后，几乎没有化学位移扰动非共价复合物的形成，而丙氨酸在P4的扰动，大概是通过与结合胰蛋白酶的活性位点的相互作用。因此，来自丝氨酸蛋白酶抑制剂的身体和反应中心环的报告表明，非共价复合物的形成不涉及丝氨酸蛋白酶抑制剂的身体中的构象变化和接触蛋白酶的区域中的反应中心环的仅微小扰动。因此，尽管丝氨酸蛋白酶抑制剂和丝氨酸蛋白酶抑制剂-蛋白酶复合物的大尺寸，分别为45和69 kDa，NMR提供了探测丝氨酸蛋白酶抑制剂构象和流动性的非常灵敏的手段，这应该适用于与一系列不同蛋白酶的非共价和共价复合物，并且可能适用于其他丝氨酸蛋白酶抑制剂。
A structural understanding of the nature and scope of serpin inhibition mechanisms has been limited by the inability so far to crystallize any serpin-proteinase complex. We describe here the application of [(1)H-(15)N]-HSQC NMR on uniformly and residue-selectively (15)N-labeled serpin alpha(1)-proteinase inhibitor (Pittsburgh variant with stabilizing mutations) to provide a nonperturbing and exquisitely sensitive means of probing the conformation of the serpin alone and in a noncovalent complex with inactive, serine 195-modified, bovine trypsin. The latter should be a good model both for the few examples of reversible serpin-proteinase complexes and for the initial Michaelis-like complex formed en route to irreversible covalent inhibition. Cleavage of the reactive center loop, with subsequent insertion into beta-sheet A, caused dramatic perturbation of most of the NMR cross-peaks. This was true for both the uniformly labeled and alanine-specifically labeled samples. The spectra of uniformly or leucine- or alanine-specifically labeled alpha(1)-proteinase inhibitor in noncovalent complex with unlabeled inactive trypsin gave almost no detectable chemical shift changes of cross-peaks, but some general increase in line width. Residue-specific assignments of the four alanines in the reactive center loop, at P12, P11, P9, and P4, allowed specific examination of the behavior of the reactive center loop. All four alanines showed higher mobility than the body of the serpin, consistent with a flexible reactive center loop, which remained flexible even in the noncovalent complex with proteinase. The three alanines near the hinge point for insertion showed almost no chemical shift perturbation upon noncovalent complex formation, while the alanine at P4 was perturbed, presumably by interaction with the active site of bound trypsin. Reporters from both the body of the serpin and the reactive center loop therefore indicate that noncovalent complex formation involves no conformational change in the body of the serpin and only minor perturbation of the reactive center loop in the region which contacts proteinase. Thus, despite the large size of serpin and serpin-proteinase complex, 45 and 69 kDa respectively, NMR provides a very sensitive means of probing serpin conformation and mobility, which should be applicable both to noncovalent and to covalent complexes with a range of different proteinases, and probably to other serpins.