Topology Scanning and Putative Three-Dimensional Structure of the Extracellular Binding Domains of the Apical Sodium-Dependent Bile Acid Transporter (SLC10A2)

Abstract

The apical sodium-dependent bile acid transporter (ASBT, SLC10A2) facilitates the enterohepatic circulation of bile salts and plays a key role in cholesterol metabolism. The membrane topology of ASBT was initially scanned using a consensus topography analysis that predominantly predicts a seven transmembrane (TM) domain configuration adhering to the "positive inside" rule. Membrane topology was further evaluated and confirmed by N-glycosylation-scanning mutagenesis, as reporter sites inserted in the putative extracellular loops 1 and 3 were glycosylated. On the basis of a 7TM topology, we built a three-dimensional model of ASBT using an approach of homology-modeling and remote-threading techniques for the extramembranous domains using bacteriorhodopsin as a scaffold for membrane attachment points; the model was refined using energy minimizations and molecular dynamics simulations. Ramachandran scores and other geometric indicators show that the model is comparable in quality to the crystal structures of similar proteins. Simulated annealing and docking of cholic acid, a natural substrate, onto the protein surface revealed four distinct binding sites. Subsequent site-directed mutagenesis of the predicted binding domain further validated the model. This model agrees further with available data for a pathological mutation (P290S) because the mutant model after in silico mutagenesis loses the ability to bind bile acids.

The solute carrier family SLC10: more than a family of bile acid transporters regarding function and phylogenetic relationships

“…However, there is still controversy about the exact number of transmembrane helices. Using N-glycosylation-scanning mutagenesis, Zhang and coworkers strongly suggested a model with seven transmembrane domains for human ASBT (Zhang et al 2004). In contrast, a nine-transmembrane domain arrangement was found by Hallén and coworkers for human ASBT and NTCP (Hallén et al 1999(Hallén et al , 2000(Hallén et al , 2002b.…”

“…Membrane insertion and sorting of NTCP and ASBT Based on bioinformatic predictions and experimental data, it has been shown that NTCP and ASBT have an extracellular N-terminus, an odd number of transmembrane helices (seven or nine) and a cytoplasmic Cterminus (Hagenbuch et al 1991;Hagenbuch and Meier 1994;Stieger et al 1994;Dawson and Oelkers 1995;Hallén et al 1999;Hallén et al 2002b;Zhang et al 2004). Several potential N-glycosylation sites are present in NTCP, ASBT and SOAT proteins, and site-directed mutagenesis has revealed that only N 5 and N 11 in rat Ntcp (Hagenbuch 1997), and N 10 in human ASBT (Zhang et al 2004) are glycosylated.…”

“…Several potential N-glycosylation sites are present in NTCP, ASBT and SOAT proteins, and site-directed mutagenesis has revealed that only N 5 and N 11 in rat Ntcp (Hagenbuch 1997), and N 10 in human ASBT (Zhang et al 2004) are glycosylated. These findings clearly placed the N-terminus of these proteins to the extracellular surface of the plasma membrane.…”

“…Vice versa, these amino acids cannot be directly involved in specific ligand binding on NTCP and ASBT, but may well affect essential protein structures and folding requirements. Using a computer-generated 3D structure model of human ASBT, Zhang and coworkers suggested a putative ligand-binding site for bile acids to the surface structure of human ASBT (Zhang et al 2004). The model identified E 282 and L 283 to be involved in the formation of dynamic hydrogen bonds with the 12α-hydroxy group of the bile acid, whereas residues in the vicinity of N 2 were capable of forming dynamic hydrogen bonds with either the 3α-or the 7α-hydroxy group of the bile acid (Zhang et al 2004).…”

“…Using a computer-generated 3D structure model of human ASBT, Zhang and coworkers suggested a putative ligand-binding site for bile acids to the surface structure of human ASBT (Zhang et al 2004). The model identified E 282 and L 283 to be involved in the formation of dynamic hydrogen bonds with the 12α-hydroxy group of the bile acid, whereas residues in the vicinity of N 2 were capable of forming dynamic hydrogen bonds with either the 3α-or the 7α-hydroxy group of the bile acid (Zhang et al 2004). However, this in silico-generated model does not fit exactly with experimental data concerning the 3D QSAR pharmacophore of ASBT (Bahringhaus et al 1999) and the localisation of the bile acid-binding site of rabbit Asbt (Kramer et al 2001a,b).…”