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An engineered disulfide bridge traps and validates an outward‐facing conformation in a bile acid transporter
Acta Crystallographica Section D ( IF 2.6 ) Pub Date : 2021-01-06 , DOI: 10.1107/s205979832001517x
Xiaodong Wang 1 , Ying Lyu 1 , Yujia Ji 1 , Ziyi Sun 1 , Xiaoming Zhou 1
Affiliation  

Apical sodium‐dependent bile acid transporter (ASBT) mediates the uptake of bile acids from the ileum lumen into enterocytes and presents a potential target for the treatment of several metabolic diseases, including type 2 diabetes. It has been proposed that the underlying mechanism for transport by ASBT is an elevator‐style alternating‐access model, which was deduced mainly by comparing high‐resolution structures of two bacterial ASBT homologs (ASBTNM from Neisseria meningitides and ASBTYf from Yersinia frederiksenii) in different conformations. However, one important issue is that the only outward‐facing structure (PDB entry 4n7x) was obtained with an Na+‐binding site mutant of ASBTYf, which severely cripples its transport function, and therefore the physiological relevance of the conformation in PDB entry 4n7x requires further careful evaluation. Here, another crystal structure is reported of ASBTYf that was captured in a state closely resembling the conformation in PDB entry 4n7x using an engineered disulfide bridge. The introduced cysteine mutations avoided any proposed Na+‐ or substrate‐binding residues, and the resulting mutant retained both structural and functional integrity and behaved similarly to wild‐type ASBTYf. These data support the hypothesis that the PDB entry 4n7x‐like structure represents a functional outward‐facing conformation of ASBTYf in its transport cycle.

中文翻译:

设计的二硫键捕获并验证胆汁酸转运蛋白中的外向构象

顶端钠依赖性胆汁酸转运蛋白 (ASBT) 介导胆汁酸从回肠腔吸收到肠细胞中,是治疗多种代谢疾病(包括 2 型糖尿病)的潜在靶点。有人提出 ASBT 运输的潜在机制是电梯式交替访问模型,这主要是通过比较两种细菌 ASBT 同源物(来自脑膜炎奈瑟菌的ASBT NM和来自耶尔森氏菌的ASBT Yf)的高分辨率结构推导出来的。在不同的构象。然而,一个重要的问题是唯一的外向结构(PDB entry 4n7x)是通过ASBT Yf的 Na +结合位点突变体获得的,这严重削弱了其运输功能,因此 PDB 条目 4n7x 中构象的生理相关性需要进一步仔细评估。在这里,报道了另一种晶体结构的 ASBT Yf,它使用工程二硫键以与 PDB 条目 4n7x 中的构象非常相似的状态捕获。引入的半胱氨酸突变避免了任何提议的 Na + - 或底物结合残基,并且由此产生的突变体保留了结构和功能的完整性,并且表现得与野生型 ASBT Yf相似。这些数据支持这样的假设,即 PDB 条目 4n7x 样结构代表了 ASBT Yf在其运输周期中的功能性外向构象。
更新日期:2021-01-06
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