Laboratorio Nacional de Genómica para la Biodiversidad, Centro de Investigación y de Estudios Avanzados del IPN, Apartado Postal 629, Irapuato, Guanajuato, CP 36821, México.
Departamento de Bioquímica y Biología Estructural, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Ciudad Universitaria, Apartado Postal 70-243, Mexico City, 04510, México.
Proteins. 2018 Jul;86(7):802-812. doi: 10.1002/prot.25519. Epub 2018 May 6.
Antibodies recognize protein targets with great affinity and specificity. However, posttranslational modifications and the presence of intrinsic disulfide-bonds pose difficulties for their industrial use. The immunoglobulin fold is one of the most ubiquitous folds in nature and it is found in many proteins besides antibodies. An example of a protein family with an immunoglobulin-like fold is the Cysteine Protease Inhibitors (ICP) family I42 of the MEROPs database for protease and protease inhibitors. Members of this protein family are thermostable and do not present internal disulfide bonds. Crystal structures of several ICPs indicate that they resemble the Ig-like domain of the human T cell co-receptor CD8α As ICPs present 2 flexible recognition loops that vary accordingly to their targeted protease, we hypothesize that members of this protein family would be ideal to design peptide aptamers that mimic protein-protein interactions. Herein, we use an ICP variant from Entamoeba histolytica (EhICP1) to mimic the interaction between p53 and MDM2. We found that a 13 amino-acid peptide derived from p53 can be introduced in 2 variable loops (DE, FG) but not the third (BC). Chimeric EhICP1-p53 form a stable complex with MDM2 at a micromolar range. Crystal structure of the EhICP1-p53(FG)-loop variant in complex with MDM2 reveals a swapping subdomain between 2 chimeric molecules, however, the p53 peptide interacts with MDM2 as in previous crystal structures. The structural details of the EhICP1-p53(FG) interaction with MDM2 resemble the interaction between an antibody and MDM2.
抗体能特异性、高亲和力地识别蛋白靶标。然而，翻译后修饰和内在二硫键的存在给它们的工业应用带来了困难。免疫球蛋白折叠是自然界中最普遍的折叠之一，除了抗体，它还存在于许多蛋白质中。免疫球蛋白样折叠蛋白家族的一个例子是半胱氨酸蛋白酶抑制剂（Cysteine Protease Inhibitors，简称 CPI）家族 I42，它是 MEROPS 蛋白酶和蛋白酶抑制剂数据库中的一个家族。该蛋白家族的成员具有热稳定性，且不存在内部二硫键。几个 CPI 的晶体结构表明，它们类似于人类 T 细胞共受体 CD8α 的 Ig 样结构域。由于 CPI 呈现出 2 个灵活的识别环，这些环会根据其靶向的蛋白酶而发生变化，因此我们假设该蛋白家族的成员将是设计模拟蛋白-蛋白相互作用的肽适体的理想选择。在此，我们使用来自溶组织内阿米巴（Entamoeba histolytica）的 CPI 变体（EhICP1）来模拟 p53 和 MDM2 之间的相互作用。我们发现，源自 p53 的 13 个氨基酸肽可以被引入到 2 个可变环（DE、FG）中，但不能引入到第三个环（BC）中。源自 p53 的 13 个氨基酸肽可以被引入到 2 个可变环（DE、FG）中，但不能引入到第三个环（BC）中。嵌合 EhICP1-p53 以微摩尔范围与 MDM2 形成稳定的复合物。与 MDM2 形成复合物的 EhICP1-p53（FG）-loop 变体的晶体结构揭示了 2 个嵌合分子之间亚结构域的交换，然而，p53 肽与 MDM2 的相互作用与之前的晶体结构相同。EhICP1-p53 与 MDM2 相互作用的结构细节类似于抗体与 MDM2 的相互作用。
