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

摘要

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 (ASBT_NM from Neisseria meningitides and ASBT_Yf 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 ASBT_Yf, 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 ASBT_Yf 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 ASBT_Yf. These data support the hypothesis that the PDB entry 4n7x-like structure represents a functional outward-facing conformation of ASBT_Yf in its transport cycle.