Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT

Abstract

High cholesterol levels greatly increase the risk of cardiovascular disease. About 50 per cent of cholesterol is eliminated from the body by its conversion into bile acids. However, bile acids released from the bile duct are constantly recycled, being reabsorbed in the intestine by the apical sodium-dependent bile acid transporter (ASBT, also known as SLC10A2). It has been shown in animal models that plasma cholesterol levels are considerably lowered by specific inhibitors of ASBT, and ASBT is thus a target for hypercholesterolaemia drugs. Here we report the crystal structure of a bacterial homologue of ASBT from Neisseria meningitidis (ASBT(NM)) at 2.2 Å. ASBT(NM) contains two inverted structural repeats of five transmembrane helices. A core domain of six helices harbours two sodium ions, and the remaining four helices pack in a row to form a flat, 'panel'-like domain. Overall, the architecture of the protein is remarkably similar to the sodium/proton antiporter NhaA, despite having no detectable sequence homology. The ASBT(NM) structure was captured with the substrate taurocholate present, bound between the core and panel domains in a large, inward-facing, hydrophobic cavity. Residues near this cavity have been shown to affect the binding of specific inhibitors of human ASBT. The position of the taurocholate molecule, together with the molecular architecture, suggests the rudiments of a possible transport mechanism.

Fig. 1. Sodium-dependent transport of bile acid by ASBT_NM

a, Time-dependent uptake of [³H]-taurocholate after expression of ASBT_NM in E. coli as monitored in buffer containing 137 mM sodium (filled circles) or <1 mM sodium (non-filled circles) b, Michaelis-Menten transport kinetics of ASBT_NM-mediated [³H]-taurocholate uptake. The Specific uptake (filled circles) was calculated by subtracting the internalization measured from control cells lacking the transporter (non-filled squares) from the total uptake (non-filled circles), as detailed in Methods. c, ASBT_NM-mediated [³H]-taurocholate uptake after 5 min in the presence of 150 μM of taurocholate, cyclosporin A, fluvastatin or bromosulfophthalein (black-filled bars) measured as a percentage of the uptake without their addition (non-filled bar). d, ASBT_NM-mediated [³H]-taurocholate uptake after 5 min for wild-type (non-filled bar) and single alanine point mutants (filled-bars): Q77A, E260A, N265A and N295A. The uptake for the mutants is displayed as a percentage of the wild type activity. The expression and detergent-solubilised folded-state of all mutants was similar to wild-type protein, Supplementary Fig. 2a. In all experiments errors bars, s.e.m.; n = 3.

Fig. 2. ASBT_NM structure

a, Ribbon representation of ASBT_NM as viewed in the plane of the membrane. TMs 1 to 10 have been coloured from red at the N-terminus to blue at the C-terminus and the position of the membrane is depicted in grey. The pink circles indicate sodium sites, Na1/Na2, and the wine-red stick model the substrate taurocholate. b, ASBT_NM structure as viewed from the intracellular side as a ribbon representation (left) and as a simplified cartoon (right): sodium ions (pink spheres), taurocholate stick model (wine red).

Fig. 3. ASBT_NM structure is inward-facing and contains bound sodium and bile acid

a, Surface representation showing the location of the taurocholate-bound intracellular cavity as a section through the protein. b, The sodium binding sites in ASBT_NM. Na1 is octahedrally coordinated by Ser114 and Asn115 on TM4b, Thr132, and Ser128 on TM5 and Glu260 on TM9a. The square pyramidal arrangement of the Na2 ligands is made up of Glu260, Val261, Met263 and Gln264 on TM9, and Gln77 on TM3. c, The intracellular cavity in ASBT_NM. Residues lining the cavity and near to the taurocholate are shown. The figures have been coloured as in Fig. 2. A 150-fold difference in inhibition of the mouse and human forms of ASBT by benzothiazepines has been assigned to sequence differences corresponding to Ser291 at the bottom of the cavity. Supplementary Figure 10 shows a stereo version of b and c.

Fig. 4. Putative mechanism for ASBT_NM transport

a, Superposition of ASBT_NM (red Panel, blue Core) and the outward-facing model as described in the text (light grey). The superposition has been optimized on the Core domains. Loops have been removed for clarity. In the image on the right the Panel of the model has been rotated 25° relative to the Core domain, around the axis shown in the left image, to superimpose the Panels. Significant kinks in the helices are represented as breaks. The area of the cavity is depicted by a salmon trapezoid. b, NhaA shown in the same view as ASBT_NM in a. The Core domain is shown in light blue and the Panel in brown. The two additional TMs and β-strands that are not present in ASBT_NM are shown in grey. The position that sodium is thought to bind is shown with a black ring. c, Schematic of the proposed mechanism that illustrates the movement of the Panel against the Core domain to transport sodium and bile acid.