title: Identification of the bile acid-binding site of the ileal lipid-binding protein by photoaffinity labeling, matrix-assisted laser desorption ionization-mass spectrometry, and NMR structure.
abstract: The ileal lipid-binding protein (ILBP) is the only physiologically relevant bile acid-binding protein in the cytosol of ileocytes. To identify the bile acid-binding site(s) of ILBP, recombinant rabbit ILBP photolabeled with 3-azi- and 7-azi-derivatives of cholyltaurine was analyzed by a combination of enzymatic fragmentation, gel electrophoresis, and matrix-assisted laser desorption ionization (MALDI)-mass spectrometry. The attachment site of the 3-position of cholyltaurine was localized to the amino acid triplet His(100)-Thr(101)-Ser(102) using the photoreactive 3,3-azo-derivative of cholyltaurine. With the corresponding 7,7-azo-derivative, the attachment point of the 7-position could be localized to the C-terminal part (position 112-128) as well as to the N-terminal part suggesting more than one binding site for bile acids. By chemical modification and NMR structure of ILBP, arginine residue 122 was identified as the probable contact point for the negatively charged side chain of cholyltaurine. Consequently, bile acids bind to ILBP with the steroid nucleus deep inside the protein cavity and the negatively charged side chain near the entry portal. The combination of photoaffinity labeling, enzymatic fragmentation, MALDI-mass spectrometry, and NMR structure was successfully used to determine the topology of bile acid binding to ILBP.
authors.affiliation: Aventis Pharma Deutschland GmbH, DG Metabolic Diseases, D-65926 Frankfurt am Main, Germany. Werner.Kramer@aventis.com
authors.forename: W
authors.initials: W
authors.lastname: Kramer
authors.forename: K
authors.initials: K
authors.lastname: Sauber
authors.forename: K H
authors.initials: KH
authors.lastname: Baringhaus
authors.forename: M
authors.initials: M
authors.lastname: Kurz
authors.forename: S
authors.initials: S
authors.lastname: Stengelin
authors.forename: G
authors.initials: G
authors.lastname: Lange
authors.forename: D
authors.initials: D
authors.lastname: Corsiero
authors.forename: F
authors.initials: F
authors.lastname: Girbig
authors.forename: W
authors.initials: W
authors.lastname: König
authors.forename: C
authors.initials: C
authors.lastname: Weyland
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001647:Bile Acids and Salts / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002756:Cholagogues and Choleretics / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006801:Humans; D015151:Immunoblotting; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D029363:Organic Anion Transporters, Sodium-Dependent; D010658:Phenylglyoxal / Q000494:pharmacology; D019920:Photoaffinity Labels / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D027981:Symporters; D013656:Taurocholic Acid / Q000737:chemistry; D013912:Threonine / Q000737:chemistry
chemical_list: D001647:Bile Acids and Salts; D002352:Carrier Proteins; D002756:Cholagogues and Choleretics; D029363:Organic Anion Transporters, Sodium-Dependent; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D027981:Symporters; C071908:sodium-bile acid cotransporter; D013912:Threonine; D012694:Serine; D006639:Histidine; D013656:Taurocholic Acid; D001120:Arginine; D010658:Phenylglyoxal