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.

Structural Requirements for Cooperativity in Ileal Bile Acid-binding Proteins

“…The binding features of BABPs appear difficult to capture, although recent studies have set important milestones in the atomic level description of BABP-ligand interactions (7)(8)(9)(10). It has been definitively ascertained that BABPs can form at least ternary complexes by hosting two ligand molecules inside a large protein cavity (11)(12)(13)(14).…”

“…Cooperativity often arises from allosteric communication, a phenomenon that frequently escapes detection by common biochemical approaches (16). NMR and calorimetric studies performed on hI-BABP and on a series of mutants have attempted to identify the communication pathways between the two binding sites (9,10). Further interaction studies reported for other members of the BABP family describe the binding stoichiometry and affinity of the rabbit ileal protein (rI-BABP) (17) as well as the binding thermodynamics and crystallographic structure of fully complexed zebrafish ileal protein (zI-BABP) (13).…”

“…Some thermodynamic binding data have been presented in the literature for human (9,11), rabbit (17), zebrafish (13), and chicken ileal BABPs (19), which allowed us to place these proteins in an indicative cooperativity scale as mentioned in the Introduction. Because it was expected that the most and the least cooperative proteins would present the largest functional differences, we focused on the structural comparison between cI-BABP (low cooperativity) and hI-BABP (high cooperativity).…”

“…The nature and orientation of residue 99 may thus have an effect on the binding mechanism. Indeed, calorimetry data coupled to mutation experiments reported for the human protein indicate that the mutant Q99A exhibits a strong decrease in cooperativity for binding to GCDA (9).…”

Síntesis de Derivados Fotoactivos de Ácidos Biliares para Aplicaciones Biológicas

“…[33] Tal como se había mencionado para el caso de los hepatocitos, es muy poco probable que los BAs atraviesen los enterocitos como monómeros libres, y el transporte intracelular es mediado por la proteína intestinal de unión a los ácidos biliares (IBABP), [34] una proteína pequeña que se encuentra citoplasmáticamente ligada al ASBT y que une los BAs (en proporción BA:IBABP 2:1) inmediatamente después de su entrada a la célula. [35] Finalmente el eflujo de BAs desde el enterocito a la sangre portal es mediado por la proteína transportadora heteromérica de solutos (OST).…”