Bile acids are synthesized from cholesterol in hepatocytes and secreted through the biliary tract into the small intestine, where they aid in absorption of lipids and fat-soluble vitamins. Through a process known as enterohepatic recirculation, more than 90% of secreted bile acids are then retrieved from the intestine and returned to the liver for resecretion1. In humans, there are two Na+-dependent bile acid transporters involved in enterohepatic recirculation, the Na+-taurocholate co-transporting polypeptide (NTCP; also known as SLC10A1) expressed in hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT; also known as SLC10A2) expressed on enterocytes in the terminal ileum2. In recent years, ASBT has attracted much interest as a potential drug target for treatment of hypercholesterolaemia, because inhibition of ASBT reduces reabsorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption3,4. However, a lack of three-dimensional structures of bile acid transporters hampers our ability to understand the molecular mechanisms of substrate selectivity and transport, and to interpret the wealth of existing functional data2,5,6,7,8. The crystal structure of an ASBT homologue from Neisseria meningitidis (ASBTNM) in detergent was reported recently9, showing the protein in an inward-open conformation bound to two Na+ and a taurocholic acid. However, the structural changes that bring bile acid and Na+ across the membrane are difficult to infer from a single structure. To understand the structural changes associated with the coupled transport of Na+ and bile acids, here we solved two structures of an ASBT homologue from Yersinia frederiksenii (ASBTYf) in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives the conserved ‘crossover’ region, where two discontinuous helices cross each other, alternating accessibility from either side of the cell membrane. This result has implications for the location and orientation of the bile acid during transport, as well as for the translocation pathway for Na+.
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Data for this study were collected at beamlines 8.2.2 at the Advanced Light Source, X-29 at the National Synchrotron Light Source, and 24ID-E and 17ID-B at the Advanced Photon Source. This work was supported by the US National Institutes of Health (R01DK088057, R01GM098878, U54GM095315 and U54GM087519), the American Heart Association (12EIA8850017), and the Cancer Prevention and Research Institute of Texas (R12MZ). M.Z. thanks R. MacKinnon for advice and guidance on scientific directions.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Purification and functional characterization of wild-type and Na+-site-mutant ASBTYf.
a, The elution profiles of wild-type (WT), E254A, Q258A and E254A/Q258A ASBTYf from a size-exclusion column. Inset shows SDS–PAGE gel of fast-performance liquid chromatography (FPLC)-purified wild-type ASBTYf before (lane 2) and after (lane 3) cleavage of the affinity tag with TEV protease. b, Chemical structures of bile acids. The primary bile acid cholic acid (top) contains a steroid nucleus, with a five-carbon side chain terminating in a carboxylic acid attached to carbon 17. Further modification of cholic acid by attachment of the amino acid taurine to the side chain results in the conjugated bile acid TCA (bottom). Both structures are oriented with the β-face towards the viewer and the α-face away from the viewer. c–e, Time courses of 1 µM 3H-TCA (20 Ci mmol−1) uptake into proteoliposomes reconstituted with wild-type (red) or E254A (blue) ASBTYf, or control liposomes (black) without protein, in the presence of 100 mM external NaCl. Uptake was measured under three conditions: in intact liposomes with an inwardly directed Na+ gradient (c); in the presence of 25 µg ml−1 of the Na+-selective ionophore gramicidin, collapsing the Na+ gradient (d); and in the presence of 25 µg ml−1 gramicidin and 0.05% of the detergent n-dodecyl-β-D-maltopyranoside (e). Under the latter condition, the liposomes are permeabilized, and only 3H-TCA bound to the lipids and protein is measured.
A schematic of the membrane topology of ASBTYf, oriented with the periplasm on top. The helices are grouped by domain, and the blue and yellow trapezoids denote transmembrane helices in the first and second inverted repeats, respectively. Pseudosymmetry-equivalent transmembrane helices are coloured identically.
a–d, Stereo images of the residues forming Na1 (a, c) and Na2 (b, d) in the ASBTNM (a, b) and ASBTYf (c, d) structures, shown with the 2Fo − Fc electron density maps in blue and the Fo − Fc density maps in green. Contour levels are set at 1.5 and 3.0σ, respectively, and the sodium ions were omitted from the Fo − Fc map calculation for the ASBTNM structure. The purple spheres in all four images correspond to the positions of Na+ in the ASBTNM structure.
Sequence alignments of human (h) NTCP, ASBT and bacterial homologues from N. meningitidis and Y. frederiksenii were calculated with CLUSTALW. The coloured bars mark the locations of transmembrane helices in ASBTYf. Residues forming Na1 and Na2 are highlighted with orange and pink, respectively. Residues in ASBTYf mutated to cysteine for the accessibility experiments are coloured green; native cysteines that were mutated to serine to make the cysteine-free background are coloured cyan.
Extended Data Figure 5 Na+-induced conformational changes in the Na+-binding sites and crossover region.
a, b, Stereoimages of the Na+-binding sites Na1 (a) and Na2 (b) are shown in the superposed ASBTYf (light blue) and ASBTNM (black) structures. Purple spheres correspond to the sodium ions in the ASBTNM structure. c, The ASBTYf structure coloured by domain, with the locations of the Na+-binding sites from ASBTNM marked with circles. Green dots mark a solvent-accessible invagination in the surface of the core domain. TM1 is hidden for clarity. d, Closer view of Na1, formed by residues from helices TM4, TM5 and TM9 of the core domain, shown in the overlaid ASBTYf (dark blue) and ASBTNM (black) structures, as viewed from periplasmic side. The purple sphere corresponds to the Na+ position in the ASBTNM structure. Green dots mark a solvent-accessible invagination in the surface of the core domain leading to the central cavity in ASBTYf, which is blocked by the residue equivalent to N109 in the Na+-bound ASBTNM structure.
Extended Data Figure 6 The core domain of ASBTYf moves relative to the membrane to form the outward-open state.
a, If the inward-open and outward-open ASBTYf structures are aligned on the core domain only (grey), a rigid motion of the panel domain (blue) moves the amphipathic helices (red) out of the inferred bilayer–periplasm and bilayer–cytoplasm interfaces. b, If the inward-open and outward-open ASBTYf structures are aligned on the panel domain only (grey), a rigid-body motion of the core domain (blue) leaves the amphipathic helices largely unaffected.
Extended Data Figure 7 Accessibility of residues in the crossover region and potential substrate-binding sites.
a, Empty liposomes or proteoliposomes reconstituted with 1:100 (mg:mg) wild-type, C196S/C248S/T106C, C196S/C248S/V123C or C196S/C248S/I269C ASBTYf were assayed for uptake of 1 µM 3H-TCA (10 Ci mmol−1) in the presence of 100 mM NaCl for the indicated time periods. b, Accessibility of the T106C, V123C and I269C residues to modification by mPEG-Mal-5K, assessed by a shift in mobility on a Coomassie-blue-stained SDS–PAGE gel (same as in Fig. 2d, shown here uncropped). Each cysteine mutant was overexpressed in E. coli and subjected to four different conditions before purification: no addition of mPEG-Mal-5K; addition of mPEG-Mal-5K to the outside of whole cells; addition of mPEG-Mal-5K after sonication to rupture the cell membranes; and addition of mPEG-Mal-5K to whole cells after block of cysteines with NEM. c, The core domain of ASBTYf, viewed from the central-cavity-facing side, with the inward accessible, outward accessible, and dual accessible surface areas coloured as in Fig. 2c. A molecule of TCA is shown modelled into two potential binding sites: left, the binding site observed in the ASBTNM structure; and right, a laterally oriented binding site based on the location of residues accessible to solution in both the inward-open and outward-open ASBTYf crystal structures. d, Surface representations of the core and panel domains of ASBTYf, both oriented with the cavity-facing sides in front, coloured by element. Carbon atoms are shown as blue-grey, oxygen atoms as red, nitrogens as dark blue, and sulphurs as yellow. e, Locations of polar residues near the crossover region. TCA is shown based on the ASBTNM structure (left) and accessibility in the ASBTYf structures (right). f, Binding of 1 µM 3H-TCA in the presence of 150 mM NaCl by wild-type and mutant ASBTYf measured with the SPA. Mutations that reduce binding by more than 20% relative to the wild-type protein are labelled in red. Error bars are s.e.m. of triplicate measurements.
a, Cartoon representation of the NapA structure (PDB accession 4BWZ) shown from two perpendicular directions. The transmembrane helices are coloured in pseudosymmetry-related pairs according to the same scheme used for the ASBT fold in Extended Data Fig. 2. Helices in the interface domain with no equivalent in the ASBT fold are coloured grey. ASBTYf is shown in the two rightmost panels for comparison. b, Outward-open (PDB accession 4BWZ, left) and inward-open (PDB accession 1ZCD, right) structures of Na+/H+ antiporters with the mobile core domain coloured dark blue and the immobile interface domain coloured red. c, Outward-open (left) and inward-open (right) structures of ASBTYf with the mobile core domain coloured dark blue and the immobile panel domain coloured red. d, Outward-open (PDB accession 1XFH, left), intermediate (PDB accession 3V8G (ref. 40), middle), and inward-open (PDB accession 3KBC, right) structures of GltPh with the mobile substrate-binding domain coloured dark blue and the immobile interface domain coloured red. e, Outward-open (PDB accession 4GBY, left), partially inward-open (PDB accession 4JA3, middle), and inward-open (PDB accession 4JA4, right) structures of E. coli XylE with the mobile C-terminal domain coloured dark blue and the immobile N-terminal domain coloured red. f, g, The core domains of ASBTYf (f) and NapA (g) are shown viewed from the side facing the panel domain, with magnified views of the crossover regions. Polar and charged residues stabilizing the exposed backbone atoms in the unwound regions are shown as sticks in both structures. The grey circles correspond to the Na+-binding sites in ASBTNM or to the approximate location of the putative Na+ binding site in NapA.
a, Location of the bound citrate molecule in the wild-type ASBTYf structure. The green surface corresponds to the Fo − Fc omit density for the citrate, contoured at 3.0σ. Helix TM1 is hidden for clarity. b, Specific binding of 0.48 µM [22Na]Cl (5.92 Ci mmol−1) to wild-type ASBTYf measured by SPA in the presence and absence of 5 mM potassium citrate. Data are from a representative experiment performed in parallel, and data points represent the mean ± s.e.m. of triplicate measurements. c, A close-up stereo-view of the area marked with a black rectangle in panel a. Likelihood-weighted 2Fo − Fc (1.5σ) and Fo − Fc (3.0σ) electron density is shown as blue and green mesh, respectively. The citrate molecule was omitted from the Fo − Fc map calculation. Potential hydrogen bonds to the protein and ordered solvent molecules are marked with dotted lines.
Animation showing the conversion between the inward-open and outward-open states in ASBTYf by linear interpolation between the WT and E254A structures, aligned on their panel domains. The core domain is colored blue, and the panel domain red. Intermediate states were calculated using the program LSQMAN. (MOV 2245 kb)
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Zhou, X., Levin, E., Pan, Y. et al. Structural basis of the alternating-access mechanism in a bile acid transporter. Nature 505, 569–573 (2014). https://doi.org/10.1038/nature12811
Nature Communications (2022)
Nature Communications (2022)