title: Determinants of cooperativity and site selectivity in human ileal bile acid binding protein.
abstract: Human ileal bile acid binding protein (I-BABP) is a member of the family of intracellular lipid-binding proteins and is thought to play a role in the enterohepatic circulation of bile salts. Our group has previously shown that human I-BABP binds two molecules of glycocholate (GCA) with low intrinsic affinity but an extraordinary high degree of positive cooperativity. Besides the strong positive cooperativity, human I-BABP exhibits a high degree of site selectivity in its interactions with GCA and glycochenodeoxycholate (GCDA), the two major bile salts in humans. In this study, on the basis of our first generation nuclear magnetic resonance (NMR) structure of the ternary complex of human I-BABP with GCA and GCDA, we introduced single-residue mutations at certain key positions in the binding pocket that might disrupt a hydrogen-bonding network, a likely way of energetic communication between the two sites. Macroscopic binding parameters were determined using isothermal titration calorimetry, and site selectivity was monitored by NMR spectroscopy of isotopically enriched bile salts. According to our results, cooperativity and site selectivity are not linked in human I-BABP. While cooperativity is governed by a subtle interplay of entropic and enthalpic contributions, site selectivity appears to be determined by more localized enthalpic effects. Possible communication pathways between the two binding sites are discussed.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA. toke@chemres.hu
authors.forename: Orsolya
authors.initials: O
authors.lastname: Toke
authors.forename: John D
authors.initials: JD
authors.lastname: Monsey
authors.forename: Gregory T
authors.initials: GT
authors.lastname: DeKoster
authors.forename: Gregory P
authors.initials: GP
authors.lastname: Tochtrop
authors.forename: Changguo
authors.initials: C
authors.lastname: Tang
authors.forename: David P
authors.initials: DP
authors.lastname: Cistola
mesh_terms: D001647:Bile Acids and Salts / Q000378:metabolism*; D001665:Binding Sites; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D007082:Ileum / Q000378:metabolism*; D007553:Isotope Labeling; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
chemical_list: D001647:Bile Acids and Salts; D006913:Hydroxysteroid Dehydrogenases; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)