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Abstract：
Two cysteine proteinase inhibitors from cowpea, VuCys1 and VuCys2, were produced in E. coli ArcticExpress (DE3). The recombinant products strongly inhibited papain and chymopapain as well as the midgut proteases from Callosobruchus maculatus larvae, a bruchid that uses cysteine proteases as major digestive enzymes. Heat treatment at 100°C for up to 60min or incubation at various pH values caused little reduction in the papain inhibitory activity of both inhibitors. Moreover, minor conformational variations, as probed by circular dichroism spectroscopy, were observed after VuCys1 and VuCys2 were subjected to these treatments. The crystal structure of VuCys1 was determined at a resolution of 1.95Å, revealing a domain-swapped dimer in the asymmetric unit. However, the two lobes of the domain-swapped dimer are positioned closer to each other in VuCys1 in comparison to other similar cystatin structures. Moreover, some polar residues from opposite lobes recruit water molecules, forming a hydrogen bond network that mediates contacts between the lobes, thus generating an extended open interface. Due to the closer distance between the lobes, a small hydrophobic core is also formed, further stabilizing the folded domain-swapped dimer. These structural features might account for the extraordinary thermal and pH stability of VuCys1.
摘要：
两种来自cow豆的半胱氨酸蛋白酶抑制剂，VuCysl和VuCys2在大肠杆菌ArcticExpress(DE3)中产生。重组产物强烈抑制木瓜蛋白酶和木瓜凝乳蛋白酶以及来自Callosobruchusmaculatus幼虫的中肠蛋白酶，使用半胱氨酸蛋白酶作为主要消化酶的布鲁克斯。在100°C热处理长达60分钟或在各种pH值下孵育几乎不会降低两种抑制剂的木瓜蛋白酶抑制活性。此外，微小的构象变化，通过圆二色性光谱探测，在对VuCys1和VuCys2进行这些处理后观察到。VuCys1的晶体结构以1.95µ的分辨率确定，揭示不对称单元中的结构域交换二聚体。然而,与其他相似的胱抑素结构相比,结构域交换二聚体的两个叶在VuCys1中的位置彼此更靠近.此外，一些来自相对叶片的极性残基募集水分子，形成氢键网络，介导叶片之间的接触，从而生成扩展的开放接口。由于叶片之间的距离更近，还形成了一个小的疏水核，进一步稳定折叠的结构域交换二聚体。这些结构特征可能解释了VuCys1非凡的热稳定性和pH稳定性。
