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

Hu NJ, Iwata S, Cameron AD, Drew D - Nature (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK.

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.

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ASBTNM structure is inward-facing and contains bound sodium and bile acida, Surface representation showing the location of the taurocholate-bound intracellular cavity as a section through the protein. b, The sodium binding sites in ASBTNM. 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 ASBTNM. 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 benzothiazepines4 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.
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Figure 3: ASBTNM structure is inward-facing and contains bound sodium and bile acida, Surface representation showing the location of the taurocholate-bound intracellular cavity as a section through the protein. b, The sodium binding sites in ASBTNM. 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 ASBTNM. 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 benzothiazepines4 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.

Mentions: ASBTNM has cytoplasmic N- and C- termini, is comprised of 10 transmembrane helices (TMs) that are linked by short loops, and has overall dimensions of approximately 45 × 30 × 30Å (Figs. 2a and b and Supplementary Fig. 3). TMs 1 to 5 and TMs 6 to 10 are topologically similar but oppositely orientated in the plane of the membrane. The r.m.s.d. (root mean square deviation) after superposition of the two topology-inverted repeats is 3.7Å (Supplementary Fig. 4a and b, and see Methods). Each repeating unit is made of an N-terminal V-motif (TMs 1-2, 6-7) and a Core motif of 3 helices (TMs 3-5, 8-10) (Fig. 2, Supplementary Fig. 3 and 4). If the V and Core-motifs are superposed separately, the r.m.s.d. is lower, 2.6Å and 2.8Å respectively (Supplementary Fig. 4c). The Core motifs from each repeat form the “Core” domain, whereas the two V-motifs create a “Panel” like domain (Fig. 2b). TMs 4 and 9 in the Core domain are broken in the middle (discontinuous), and form helical hairpins with kinked TMs 5 and 10, respectively. At the point where TMs 4 and 9 are broken by well-conserved peptide motifs, they cross over (Fig. 2, Supplementary Fig. 5 and 6). On the intracellular side a wide crevice separates the Core from the Panel domain (Fig. 3a). The cavity extends over halfway through the protein. The extracellular side of the cavity is tightly closed by TMs 1, 2, 4b, 7, 9b and 10. Previously, two topology models of ASBT were proposed with 7 or 9 TMs respectively17,18. As TM1 is not conserved in ASBT the structure is broadly consistent with the 9-TM model (Supplementary Fig. 5). TMs 4 and 9 were annotated as extracellular loops in the 7-TM topology model, but were correctly identified in the 9-TM model.


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

Hu NJ, Iwata S, Cameron AD, Drew D - Nature (2011)

ASBTNM structure is inward-facing and contains bound sodium and bile acida, Surface representation showing the location of the taurocholate-bound intracellular cavity as a section through the protein. b, The sodium binding sites in ASBTNM. 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 ASBTNM. 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 benzothiazepines4 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.
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Figure 3: ASBTNM structure is inward-facing and contains bound sodium and bile acida, Surface representation showing the location of the taurocholate-bound intracellular cavity as a section through the protein. b, The sodium binding sites in ASBTNM. 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 ASBTNM. 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 benzothiazepines4 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.
Mentions: ASBTNM has cytoplasmic N- and C- termini, is comprised of 10 transmembrane helices (TMs) that are linked by short loops, and has overall dimensions of approximately 45 × 30 × 30Å (Figs. 2a and b and Supplementary Fig. 3). TMs 1 to 5 and TMs 6 to 10 are topologically similar but oppositely orientated in the plane of the membrane. The r.m.s.d. (root mean square deviation) after superposition of the two topology-inverted repeats is 3.7Å (Supplementary Fig. 4a and b, and see Methods). Each repeating unit is made of an N-terminal V-motif (TMs 1-2, 6-7) and a Core motif of 3 helices (TMs 3-5, 8-10) (Fig. 2, Supplementary Fig. 3 and 4). If the V and Core-motifs are superposed separately, the r.m.s.d. is lower, 2.6Å and 2.8Å respectively (Supplementary Fig. 4c). The Core motifs from each repeat form the “Core” domain, whereas the two V-motifs create a “Panel” like domain (Fig. 2b). TMs 4 and 9 in the Core domain are broken in the middle (discontinuous), and form helical hairpins with kinked TMs 5 and 10, respectively. At the point where TMs 4 and 9 are broken by well-conserved peptide motifs, they cross over (Fig. 2, Supplementary Fig. 5 and 6). On the intracellular side a wide crevice separates the Core from the Panel domain (Fig. 3a). The cavity extends over halfway through the protein. The extracellular side of the cavity is tightly closed by TMs 1, 2, 4b, 7, 9b and 10. Previously, two topology models of ASBT were proposed with 7 or 9 TMs respectively17,18. As TM1 is not conserved in ASBT the structure is broadly consistent with the 9-TM model (Supplementary Fig. 5). TMs 4 and 9 were annotated as extracellular loops in the 7-TM topology model, but were correctly identified in the 9-TM model.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK.

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.

Show MeSH
Related in: MedlinePlus