Title : 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