Heterodimerization of the Entamoeba histolytica EhCPADH virulence complex through molecular dynamics and protein–protein docking

EhCPADH is a protein complex involved in the virulence of Entamoeba histolytica, the protozoan responsible for human amebiasis. It is formed by the EhCP112 cysteine protease and the EhADH adhesin. To explore the molecular basis of the complex formation, three-dimensional models were built for both proteins and molecular dynamics simulations (MDS) and docking calculations were performed. Results predicted that the pEhCP112 proenzyme and the mEhCP112 mature enzyme were globular and peripheral membrane proteins. Interestingly, in pEhCP112, the propeptide appeared hiding the catalytic site (C167, H329, N348); while in mEhCP112, this site was exposed and its residues were found structurally closer than in pEhCP112. EhADH emerged as an extended peripheral membrane protein with high fluctuation in Bro1 and V shape domains. 500 ns-long MDS and protein–protein docking predictions evidenced different heterodimeric complexes with the lowest free energy. pEhCP112 interacted with EhADH by the propeptide and C-terminal regions and mEhCP112 by the C-terminal through hydrogen bonds. In contrast, EhADH bound to mEhCP112 by 442–479 residues, adjacent to the target cell-adherence region (480–600 residues), and by the Bro1 domain (9–349 residues). Calculations of the effective binding free energy and per residue free energy decomposition showed that EhADH binds to mEhCP112 with a higher binding energy than to pEhCP112, mainly through van der Waals interactions and the nonpolar part of solvation energy. The EhADH and EhCP112 structural relationship was validated in trophozoites by immunofluorescence, TEM, and immunoprecipitation assays. Experimental findings fair agreed with in silico results.

EhCPADH是一种参与溶组织内阿米巴（Entamoeba histolytica）致病过程的蛋白复合物，该原生动物是引发人类阿米巴病的病原体。它由EhCP112半胱氨酸蛋白酶与EhADH黏附素组成。为探究该复合物形成的分子基础，研究人员分别构建了两种蛋白的三维模型，并开展了分子动力学模拟（MDS）与分子对接计算。结果预测显示，pEhCP112酶原与mEhCP112成熟酶均为球状外周膜蛋白。值得注意的是，在pEhCP112中，前肽遮蔽了催化位点（C167、H329、N348）；而在mEhCP112中，该催化位点处于暴露状态，且其残基的空间距离较pEhCP112中的残基更短，构象更为紧凑。EhADH则呈现为延展型外周膜蛋白，在Bro1结构域与V形结构域中存在较高的构象波动。500纳秒时长的分子动力学模拟与蛋白-蛋白对接预测结果表明，存在多种异二聚体复合物，其中自由能最低的复合物为：pEhCP112可通过其前肽与C端区域与EhADH结合，而mEhCP112则凭借C端区域与EhADH形成氢键。与之相反，EhADH通过442-479号残基区段（紧邻靶细胞黏附区域，即480-600号残基区段）以及Bro1结构域（9-349号残基区段）与mEhCP112结合。有效结合自由能与单残基自由能分解计算结果显示，EhADH与mEhCP112的结合自由能高于与pEhCP112的结合自由能，这一差异主要由范德华相互作用与溶剂化能的非极性部分贡献。研究人员通过免疫荧光、透射电子显微镜（TEM）与免疫沉淀实验在滋养体中验证了EhADH与EhCP112的结构相互作用关系，实验结果与计算机模拟结果基本吻合。