Identification of a ligand-binding site in the Na+/Bile acid cotransporting protein from rabbit ileum

Reabsorption of bile acids occurs in the terminal ileum by a Na+-dependent transport system composed of several subunits of the ileal bile acid transp orter (IBAT) and the ileal lipid-binding protein. To identify the bile acid -binding site of the transporter protein IBAT, ileal brush border membrane vesicles from rabbit ileum were photoaffinity labeled with a radioactive 7- azi-derivative of cholyltaurine followed by enrichment of IBAT protein by p reparative SDS gel electrophoresis. Enzymatic fragmentation with chymotrypi sin yielded IBAT peptide fragments in the molecular range of 20.4-4 kDa. Wi th epitope-specific antibodies generated against the C terminus a peptide o f molecular mass of 6.6-7 kDa was identified as the smallest peptide fragme nt carrying both the C terminus and the covalently attached radio-labeled b ile acid derivative. This clearly indicates that the ileal Na+/bile acid co transporting protein IBAT contains a bile acid-binding site within the C-te rminal 56-67 amino acids. Based on the seven-transmembrane domain model for IBAT, the bile acid-binding site is localized to a region containing the s eventh transmembrane domain and the cytoplasmic C terminus. Alternatively, assuming the nine-transmembrane domain model, this bile acid-binding site i s localized to the ninth transmembrane domain and the C terminus.