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 ASBTYf, 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 ASBTYf 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 ASBTYf 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.
PDB references: ASBTYf-Pair1Linked, Y113C/P190C mutant after disulfide cross-linking, 6lh1; ASBTYf-Pair1Free, Y113C/P190C mutant before disulfide cross-linking, 7cyg; ASBTYf-Pair2Free, V110C/I197C mutant before disulfide cross-linking, 7cyk.
References
- ↑ 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
