Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 A and full insertion of the reactive center loop into beta-sheet A.
Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 A and full insertion of the reactive center loop into beta-sheet A.
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DOI：
10.1073/pnas.96.9.4808
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发表时间：
1999-04
影响因子：
11.1
通讯作者：
E. Stratikos;P. Gettins
中科院分区：
文献类型：
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作者：
E. Stratikos;P. Gettins
为了确定共价丝氨酸蛋白酶抑制剂-蛋白酶复合物中蛋白酶的位置，我们制备了丝氨酸蛋白酶抑制剂α1-蛋白酶抑制剂的匹兹堡变体的七个含单半胱氨酸的变体，并用丹酰基荧光团标记每个半胱氨酸。丹酰探针用于通过直接扰动和通过从胰蛋白酶中的色氨酸到丹酰的荧光能量转移来确定蛋白酶胰蛋白酶与丝氨酸蛋白酶抑制剂的共价和非共价复合物的接近度。仅在共价复合物中的两个位置和非共价复合物中的一个位置上观察到对丹磺酰荧光团的巨大直接影响。距离范围从 <14 A 到 64 A 被用来严格限制复合体的可能结构。与距离限制和丹酰基荧光团的直接扰动一致的结构将蛋白酶放置在距初始对接位点丝氨酸蛋白酶抑制剂的远端。蛋白酶的这一位置需要蛋白酶从丝氨酸蛋白酶抑制剂的一端完全易位到另一端，并将反应中心环完全插入β-片层A中以形成动力学捕获的复合物。随后丝氨酸蛋白酶抑制剂和蛋白酶的紧密并置可以解释蛋白酶活性位点的扭曲是如何发生的，以及因此有多少种丝氨酸蛋白酶抑制剂和蛋白酶的组合可以被常见的构象变化机制抑制。
To determine the location of the proteinase in the covalent serpin-proteinase complex we prepared seven single-cysteine-containing variants of the Pittsburgh variant of the serpin alpha1-proteinase inhibitor, and we labeled each cysteine with the dansyl fluorophore. The dansyl probes were used to determine proximity of the proteinase trypsin in covalent and noncovalent complexes with the serpin, both by direct perturbation and by fluorescence energy transfer from tryptophans in trypsin to dansyl. Large direct effects on dansyl fluorophores were seen for only two positions in covalent complex and one position in noncovalent complex. Distances ranging from <14 A to 64 A were used to severely constrain possible structures for the complex. The structure consistent with both distance constraints and direct perturbations of the dansyl fluorophores placed the proteinase at the distal end of the serpin from the initial docking site. This position for the proteinase requires complete translocation of the proteinase from one end of the serpin to the other and full insertion of the reactive center loop into beta-sheet A to form the kinetically trapped complex. The consequent tight juxtapositioning of serpin and proteinase could explain how distortion of the proteinase active site can occur and hence how many combinations of serpin and proteinase can be inhibited by a common conformational change mechanism.