Important role of the Cys-191-Cys-220 disulfide bond in thrombin function and allostery
Important role of the Cys-191-Cys-220 disulfide bond in thrombin function and allostery
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DOI：
10.1074/jbc.m703202200
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发表时间：
2007-09-14
影响因子：
4.8
通讯作者：
Di Cera, Enrico
中科院分区：
文献类型：
--
作者：
Bush-Pelc, Leslie A.;Marino, Francesca;Di Cera, Enrico
关于二硫键在丝氨酸蛋白酶催化结构域中的作用，人们知之甚少。Cys-191-Cys-220二硫键位于190链和220环之间，190链通向氧阴离子空穴，220环构成了变构蛋白酶中初级特异性口袋和Na+结合部位的结构。除去凝血酶中的这一键，对几种在P1处携带Arg或Lys的发色和自然底物的活性损失类似于100倍。Na+的激活受到抑制，并且不能检测到对Na+结合的荧光变化。C191A/C220A突变体在自由形式下的1.54埃分辨结构显示了类似于野生型的无Na+慢型的构象。缺乏二硫键使Asp-189的侧链暴露在溶剂中，翻转了Gly-219的主干原子，并在定义Na+位的186和220环的部分中产生了无序。这种构象以Na+位点的扰动为特征，但具有底物可及的活性部位，提供了最近发现的凝血酶的E*形式的可能代表。根据络合物的1.8埃分辨结构，活性位点抑制剂H-D-Phe-Pro-Arg-CH2Cl纠正了186和220环中的无序和Gly-219的翻转。我们得出结论，Cys-191-Cys-220二硫键通过屏蔽Asp-189与溶剂的作用而使初级特异性口袋变得稳定，并使Gly-219的主链O原子定向以获得最佳的底物结合。此外，二硫键稳定了186和220环，这是Na+结合和激活的关键。
Little is known on the role of disulfide bonds in the catalytic domain of serine proteases. The Cys-191-Cys-220 disulfide bond is located between the 190 strand leading to the oxyanion hole and the 220-loop that contributes to the architecture of the primary specificity pocket and the Na+ binding site in allosteric proteases. Removal of this bond in thrombin produces an similar to 100-fold loss of activity toward several chromogenic and natural substrates carrying Arg or Lys at P1. Na+ activation is compromised, and no fluorescence change can be detected in response to Na+ binding. A 1.54-angstrom resolution structure of the C191A/C220A mutant in the free form reveals a conformation similar to the Na+-free slow form of wild type. The lack of disulfide bond exposes the side chain of Asp-189 to solvent, flips the backbone Oatom of Gly-219, and generates disorder in portions of the 186 and 220 loops defining the Na+ site. This conformation, featuring perturbation of the Na+ site but with the active site accessible to substrate, offers a possible representation of the recently identified E* form of thrombin. Disorder in the 186 and 220 loops and the flip of Gly-219 are corrected by the active site inhibitor H-D-Phe-Pro-Arg-CH2Cl, as revealed by the 1.8-angstrom resolution structure of the complex. We conclude that the Cys-191-Cys-220 disulfide bond confers stability to the primary specificity pocket by shielding Asp-189 from the solvent and orients the backbone O atom of Gly-219 for optimal substrate binding. In addition, the disulfide bond stabilizes the 186 and 220 loops that are critical for Na+ binding and activation.