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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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Sodium-dependent transport of bile acid by ASBTNMa, Time-dependent uptake of [3H]-taurocholate after expression of ASBTNM 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 ASBTNM-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, ASBTNM-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, ASBTNM-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.
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Figure 1: Sodium-dependent transport of bile acid by ASBTNMa, Time-dependent uptake of [3H]-taurocholate after expression of ASBTNM 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 ASBTNM-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, ASBTNM-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, ASBTNM-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.

Mentions: ASBTNM from Neisseria meningitidis, with 26% identity and 54% similarity to human ASBT was identified by fluorescent-based screening methods10,11 as a suitable candidate for structural studies (Supplementary Fig. 1 and Fig. 2). Residues known to be functionally important in mammalian ASBT and other SLC10 members12 are well conserved in ASBTNM (Supplementary Fig. 1). Bile acid transport by ASBTNM was confirmed in whole-cells by the sodium-dependent uptake of [3H]-taurocholate (Fig. 1a). The observed Km for [3H]-taurocholate is in the low μM range ~50μM (Fig. 1b), which is similar to that measured for rat and human ASBT7,13,14. The ASBT inhibitors cyclosporin A15, bromosulfophthalein15 and the drug Fluvastatin16, are also competitors for ASBTNM-mediated [3H]-taurocholate transport (Fig. 1c). Thus, ASBTNM is a valid model of mammalian bile acid transporters. The ASBTNM structure was solved by single wavelength anomalous scattering and refined at a resolution of 2.2Å (Supplementary Tables 1 and 2, see Methods).


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

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

Sodium-dependent transport of bile acid by ASBTNMa, Time-dependent uptake of [3H]-taurocholate after expression of ASBTNM 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 ASBTNM-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, ASBTNM-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, ASBTNM-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.
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Related In: Results  -  Collection

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Figure 1: Sodium-dependent transport of bile acid by ASBTNMa, Time-dependent uptake of [3H]-taurocholate after expression of ASBTNM 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 ASBTNM-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, ASBTNM-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, ASBTNM-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.
Mentions: ASBTNM from Neisseria meningitidis, with 26% identity and 54% similarity to human ASBT was identified by fluorescent-based screening methods10,11 as a suitable candidate for structural studies (Supplementary Fig. 1 and Fig. 2). Residues known to be functionally important in mammalian ASBT and other SLC10 members12 are well conserved in ASBTNM (Supplementary Fig. 1). Bile acid transport by ASBTNM was confirmed in whole-cells by the sodium-dependent uptake of [3H]-taurocholate (Fig. 1a). The observed Km for [3H]-taurocholate is in the low μM range ~50μM (Fig. 1b), which is similar to that measured for rat and human ASBT7,13,14. The ASBT inhibitors cyclosporin A15, bromosulfophthalein15 and the drug Fluvastatin16, are also competitors for ASBTNM-mediated [3H]-taurocholate transport (Fig. 1c). Thus, ASBTNM is a valid model of mammalian bile acid transporters. The ASBTNM structure was solved by single wavelength anomalous scattering and refined at a resolution of 2.2Å (Supplementary Tables 1 and 2, see Methods).

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