Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

• Published in: Journal of molecular biology Vol. 433; no. 4; p. 166764
• Main Authors: Lu, Pei-Hua, Li, Chieh-Chin, Chiang, Yun-Wei, Liu, Jyung-Hurng, Chiang, Wesley Tien, Chao, Yi-Hsuan, Li, Guan-Syun, Weng, Shao-En, Lin, Sung-Yao, Hu, Nien-Jen
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 19.02.2021
• Subjects: alternating access; bile acid transporter; DEER spectroscopy; elevator mechanism; PEGylation; MTSSL; DEER spectroscopy; bile acid transporter; ESR; elevator mechanism; alternating access; DDM; SDAF; SEC; DEER; PEGylation; TCH; ASBT; mPEG-MAL-5K
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2020.166764

[Display omitted] •The mechanism of conformational isomerization of ASBTNM remains undefined.•Na+ binding to ASBTNM does not favor an outward-facing conformation.•DEER distance measurements are in agreement with SDAF profiles.•ASBTNM exists as a monomer in detergent micelles and membranes.•The dynamics studies provide a new viewpoint of elevator-type transporters. Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na+ as the driving force. The crystal structures of two bacterial homologues ASBTNM and ASBTYf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na+-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na+ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBTNM under different Na+ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBTNM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na+ ions shift the conformational equilibrium of ASBTNM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.