Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, Circuito exterior s/n, Coyoacán, Ciudad de Mexico 04510, Mexico.
Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, km. 9.6 Libramiento Norte Carretera Irapuato-León, CP 36821 Irapuato, Guanajuato, Mexico.
Biochim Biophys Acta Proteins Proteom. 2020 Nov;1868(11):140512. doi: 10.1016/j.bbapap.2020.140512. Epub 2020 Jul 28.
The genome of Entamoeba histolytica encodes approximately 50 Cysteine Proteases (CPs) whose activity is regulated by two Inhibitors of Cysteine Proteases (ICPs), EhICP1 and EhICP2. The main difference between both EhICPs is the acquisition of a 17 N-terminal targeting signal in EhICP2 and three exposed cysteine residues in EhICP1. The three exposed cysteines in EhICP1 potentiate the formation of cross-linking species that drive heterogeneity. Here we solved the NMR structure of EhICP1 using a mutant protein without accessible cysteines. Our structural data shows that EhICP1 adopts an immunoglobulin fold composed of seven β-strands, and three solvent exposed loops that resemble the structures of EhICP2 and chagasin. EhICP1 and EhICP2 are able to inhibit the archetypical cysteine protease papain by intercalating their BC loops into the protease active site independently of the character of the residue (serine or threonine) responsible to interact with the active site of papain. EhICP1 and EhICP2 present signals of functional divergence as they clustered in different clades. Two of the three exposed cysteines in EhICP1 are located at the DE loop that intercalates into the CP substrate-binding cleft. We propose that the solvent exposed cysteines of EhICP1 play a role in regulating its inhibitory activity and that in oxidative conditions, the cysteines of EhICP1 react to form intra and intermolecular disulfide bonds that render an inactive inhibitor. EhICP2 is not subject to redox regulation, as this inhibitor does not contain a single cysteine residue. This proposed redox regulation may be related to the differential cellular localization between EhICP1 and EhICP2.
溶组织内阿米巴的基因组大约编码 50 种半胱氨酸蛋白酶（CPs），其活性受两种半胱氨酸蛋白酶抑制剂（ICPs）EhICP1 和 EhICP2 调节。两种 EhICPs 的主要区别在于 EhICP2 获得了 17 个 N 端靶向信号，而 EhICP1 中有三个暴露的半胱氨酸残基。EhICP1 中的三个暴露的半胱氨酸有助于形成交联物种，从而导致异质性。在这里，我们使用一种没有可及半胱氨酸的突变蛋白解决了 EhICP1 的 NMR 结构。我们的结构数据表明，EhICP1 采用了由七个β-链和三个暴露溶剂的环组成的免疫球蛋白折叠，这些环类似于 EhICP2 和 chagasin 的结构。EhICP1 和 EhICP2 能够通过将其 BC 环插入蛋白酶活性位点来抑制典型的半胱氨酸蛋白酶木瓜蛋白酶，而与负责与木瓜蛋白酶活性位点相互作用的残基（丝氨酸或苏氨酸）的性质无关。EhICP1 和 EhICP2 存在功能分化的信号，因为它们聚类在不同的分支中。EhICP1 中的三个暴露半胱氨酸中有两个位于 DE 环，该环插入 CP 底物结合裂隙中。我们提出，EhICP1 中的暴露半胱氨酸在调节其抑制活性中起作用，并且在氧化条件下，EhICP1 的半胱氨酸反应形成内和分子间二硫键，从而使抑制剂失活。EhICP2 不受氧化还原调节，因为该抑制剂不含单个半胱氨酸残基。这种拟议的氧化还原调节可能与 EhICP1 和 EhICP2 之间的细胞定位差异有关。
