title: Bile acid interactions with rabbit ileal lipid binding protein and an engineered helixless variant reveal novel ligand binding properties of a versatile beta-clam shell protein scaffold.
abstract: The intracellular ileal lipid binding proteins (ILBPs) are involved in the transport and enterohepatic circulation of bile acids. ILBPs from different species show high sequence and structural homology and have been shown to bind multiple bile acid ligands with differing degrees of selectivity and positive co-operativity. Human ILBP binds bile acid derivatives in a well-characterised 2:1 ligand:protein complex, however, we show that the highly homologous rabbit ILBP (82% sequence identity) with seven conservative substitutions preferentially binds multiple conjugated deoxycholate ligands in a novel 3:1 binding mode essentially within the same beta-clam shell structure. We have extended these studies to investigate the role of the alpha-helical capping motif (residues 9-35) in controlling the dimensions of the binding cavity and ligand uptake. Substituting the alpha-helical motif (residues 9-35) with a short Gly-Gly-Ser-Gly linker dramatically affects the protein stability such that under physiological conditions the mutant (Deltaalpha-ILBP) is highly disordered. However, we show that the inability of the mutant to adopt a stable three-dimensional structure under these conditions is no barrier to binding ligands with near-native affinity. These structural modifications not only demonstrate the possibility of strong coupling between ligand binding and protein folding, but result in changes in bile acid selectivity and binding stoichiometry, which we characterise in detail using isothermal calorimetry and mass spectrometry.
authors.affiliation: Centre for Biomolecular Sciences, School of Chemistry, University Park, Nottingham NG7 2RD, UK.
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Kouvatsos
authors.forename: Victoria
authors.initials: V
authors.lastname: Thurston
authors.forename: Kathryn
authors.initials: K
authors.lastname: Ball
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Oldham
authors.forename: Neil R
authors.initials: NR
authors.lastname: Thomas
authors.forename: Mark S
authors.initials: MS
authors.lastname: Searle
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002151:Calorimetry; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D027981:Symporters / Q000737:chemistry*
chemical_list: D008024:Ligands; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; C071908:sodium-bile acid cotransporter