Structural characterization of core-bradavidin in complex with biotin

Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin–Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin.

布拉德亲和素（Bradavidin）是一种四聚体生物素结合蛋白，与鸡卵白素（chicken avidin）及细菌链霉亲和素（streptavidin）类似，最初从固氮菌慢生根瘤菌（Bradyrhizobium diazoefficiens）中克隆得到。我们此前曾报道了全长野生型（wt）布拉德亲和素的晶体结构，该蛋白包含138个氨基酸，其C端残基Gly129-Lys138（命名为"Brad-tag"）可作为内源性配体——即Gly129-Lys138可结合至同一四聚体中相邻亚基的生物素结合位点，且具备作为生物技术用亲和标签的潜力。本研究中，缺失C端残基Gly114-Lys138（即不含"Brad-tag"）的核心布拉德亲和素（core-bradavidin）与生物素的复合物晶体结构经解析，分辨率达1.60 Å，相关结构已存入PDB:4BBO。我们还报道了沼泽红假单胞菌（Rhodopseudomonas palustris）来源的类亲和素蛋白红亲和素（rhodavidin），以及慢生根瘤菌属（Bradyrhizobium sp.）Ai1a-2菌株来源的类亲和素蛋白的同源建模模型，二者的C端均带有"Brad-tag"序列。此外，原始核心布拉德亲和素的工程化变体核心布拉德亲和素V1，以及核心布拉德亲和素的双突变体（Cys39Ala与Cys69Ala，即CC突变体）均可在大肠杆菌（E. coli）中实现高效表达。本研究数据可为核心布拉德亲和素-"Brad-tag"配对体系的进一步工程化改造提供支撑，以用于生物技术检测及化学生物学应用，同时可加深我们对布拉德亲和素生物素结合模式的认知。