Journal:Acta Cryst D:S205979832001517X

An engineered disulfide bridge traps and validates an outward-facing conformation in a bile acid transporter

Xiaodong Wang, Ying Lyu, Yujia Ji, Ziyi Sun and Xiaoming Zhou ^[1]

Molecular Tour
Apical sodium-dependent bile acid transporter (ASBT) is a membrane transporter protein which recycles bile acids from small intestine into enterocytes. It is a potential drug target for treating several metabolic diseases including type 2 diabetes. To date, high-resolution structures are only available for two bacterial ASBT proteins, which provide structural information to help understand how a bile acid is transported by ASBT. To do so, a minimum of two ASBT conformational states have to be obtained, one facing outside of the cell and the other facing inside. However, currently the only outward-facing ASBT structure is obtained with a severely crippled ASBT protein, whose normal functions such as binding and transport of bile acids are nearly lost. Therefore, the outward-facing state of ASBT needs validation.

In this study, a bacterial ASBT, called ASBT_Yf, is engineered to have its intracellular parts connected by a disulfide bond, so that it will be trapped in an outward-facing state. Before this engineered ASBT_Yf is trapped, it folds and moves like the wild-type protein. More importantly, it remains functional since it binds bile acids as normal. Then after the conformational trap, this ASBT_Yf is found to open outwardly in its structure. In other words, a wild-type-like ASBT protein is trapped in a state facing outside of the cell, demonstrating that this outward-facing state is of physiological relevance. Meanwhile, a low-affinity ligand-like molecule, citrate, binds to the substrate-binding site in the trapped outward-facing ASBT structure, further indicating that the trapped ASBT protein retains its functionality. These data validate a physiological outward-facing state in ASBT, and advance out understanding toward its transport mechanism.

(PDB entry 6lh1). Solvent can access the central binding pocket from outside of the cell.

• .

(PDB entry 6lh1), indicating this protein retains functionality.

References

1. ↑ Wang X, Lyu Y, Ji Y, Sun Z, Zhou X. An engineered disulfide bridge traps and validates an outward-facing conformation in a bile acid transporter. Acta Crystallogr D Struct Biol. 2021 Jan 1;77(Pt 1):108-116. doi:, 10.1107/S205979832001517X. Epub 2021 Jan 1. PMID:33404530 doi:http://dx.doi.org/10.1107/S205979832001517X