Determinants of cooperativity and site selectivity in human ileal bile acid binding protein

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.     

Kinetic mechanism of ligand binding in human ileal bile acid binding protein as determined by stopped-flow fluorescence analysis

Cooperative ligand binding to human ileal bile acid binding protein (I-BABP) was studied using the stopped-flow fluorescence technique. The kinetic data obtained for wild-type protein are in agreement with a four-step mechanism where after a fast conformational change on the millisecond time scale, the ligands bind in a sequential manner, followed by another, slow conformational change on the time scale of seconds. This last step is more pronounced in the case of glycocholate (GCA), the bile salt that binds with high positive cooperativity and is absent in mutant I-BABP proteins that lack positive cooperativity in their bile salt binding. These results suggest that positive cooperativity in human I-BABP is related to a slow conformational change of the protein, which occurs after the second binding step. Analogous to that in the intestinal fatty acid binding protein (I-FABP), we hypothesize that ligand binding in I-BABP is linked to a disorder-order transition between an open and a closed form of the protein.     

Internal motions and exchange processes in human ileal bile acid binding protein as studied by backbone (15)N nuclear magnetic resonance spectroscopy

Human ileal bile acid binding protein (I-BABP), a member of the family of intracellular lipid binding proteins, is thought to play a role in the enterohepatic circulation of bile salts. Previously, we have shown by stopped-flow fluorescence analysis that positive binding cooperativity exhibited by I-BABP in its interactions with glycocholate (GCA) and glycochenodeoxycholate (GCDA), the two primary bile salts in humans, is related to a slow conformational change in the protein. In this study, we used backbone (15)N relaxation nuclear magnetic resonance (NMR) techniques to obtain residue-specific information about the internal dynamics of apo I-BABP and the doubly ligated I-BABP:GCA:GCDA complex on various time scales. According to our NMR data, bile salt binding is accompanied by a slight rigidification of the (15)N-(1)H bond vectors on the picosecond to nanosecond time scale, with most pronounced changes occurring in the C-D region. In contrast to the minor effects of ligation on fast motions, relaxation dispersion NMR experiments indicate a marked difference between the two protein states on the microsecond to millisecond time scale. In the apo form, an extensive network of conformational fluctuations is detected throughout segments of the EFGHIJ β-strands and the C-D loop, which cease upon complexation. Our NMR data are in agreement with a conformational selection model we proposed earlier for I-BABP and support the hypothesis of an allosteric mechanism of ligand binding. According to the NMR measurements, the helical cap region may have a less crucial role in mediating ligand entry and release than what has been indicated for fatty acid binding proteins.     

NMR-based modeling and binding studies of a ternary complex between chicken liver bile acid binding protein and bile acids

Chicken liver bile acid binding protein (cL-BABP) is involved in bile acid transport in the liver cytosol. A detailed study of the mechanism of binding and selectivity of bile acids binding proteins towards the physiological pool of bile salts is a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. In the present study, we modeled the ternary complex of cL-BABP with two molecules of bile salts using the data driven docking program HADDOCK on the basis of NMR and mass spectrometry data. Docking resulted in good 3D models, satisfying the majority of experimental restraints. The docking procedure represents a necessary step to help in the structure determination and in functional analysis of such systems, in view of the high complexity of the 3D structure determination of a ternary complex with two identical ligands. HADDOCK models show that residues involved in binding are mainly located in the C-terminal end of the protein, with two loops, CD and EF, playing a major role in ligand binding. A spine, comprising polarresidues pointing toward the protein interior and involved in motion communication, has a prominent role in ligand interaction. The modeling approach has been complemented with NMR interaction and competition studies of cL-BABP with chenodeoxycholic and cholic acids. A higher affinity for chenodeoxycholic acid was observed and a Kd upper limit estimate was obtained. The binding is highly cooperative and no site selectivity was detected for the different bile salts, thus indicating that site selectivity and cooperativity are not correlated. Differences in physiological pathways and bile salt pools in different species is discussed in light of the binding results thus enlarging the body of knowledge of BABPs biological functions.     

Characterization of a novel 14 kDa bile acid-binding protein from rat ileal cytosol

A 14 kDa polypeptide in rat ileal cytosol has been identified as the major intestinal cytosolic bile acid-binding protein (I-BABP) by photoaffinity labeling with the radiolabeled 7,7-azo derivative of taurocholate (7,7-azo-TC). To further characterize I-BABP, the protein was purified by lysylglycocholate Sepharose 4B affinity and DE-52 anion-exchange chromatography. The purified I-BABP contained a single 14 kDa band on SDS-PAGE. The 14 kDa protein showed a 26-fold increase in binding affinity for [3H]7,7-azo-TC compared to cytosolic protein. Immunoblotting of protein fractions separated by affinity chromatography showed that neither liver fatty acid binding protein (L-FABP) nor intestinal fatty acid binding protein (I-FABP) bind to the affinity column and that the 14 kDa protein which bound to the column and was subsequently eluted with detergent did not cross-react with anti-L-FABP or anti-I-FABP. The 14 kDa protein labeled with [3H]7,7-azo-TC was radioimmunoprecipitated from cytosol by rabbit antiserum raised against purified I-BABP. I-BABP was shown to have a blocked N-terminus; however, its mixed internal sequence generated from cyanogen bromide-cleaved protein and amino acid composition indicated that it was related to (although clearly distinct from) both I-FABP and L-FABP. These studies have isolated a 14 kDa bile acid-binding protein from rat ileal cytosol which is immunologically and biochemically distinct from I-FABP and L-FABP.     

Structural requirements for cooperativity in ileal bile acid-binding proteins

Ileal bile acid-binding proteins (I-BABP), belonging to the family of intracellular lipid-binding proteins, control bile acid trafficking in enterocytes and participate in regulating the homeostasis of these cholesterol-derived metabolites. I-BABP orthologues share the same structural fold and are able to host up to two ligands in their large internal cavities. However variations in the primary sequences determine differences in binding properties such as the degree of binding cooperativity. To investigate the molecular requirements for cooperativity we adopted a gain-of-function approach, exploring the possibility to turn the noncooperative chicken I-BABP (cI-BABP) into a cooperative mutant protein. To this aim we first solved the solution structure of cI-BABP in complex with two molecules of the physiological ligand glycochenodeoxycholate. A comparative structural analysis with closely related members of the same protein family provided the basis to design a double mutant (H99Q/A101S cI-BABP) capable of establishing a cooperative binding mechanism. Molecular dynamics simulation studies of the wild type and mutant complexes and essential dynamics analysis of the trajectories supported the role of the identified amino acid residues as hot spot mediators of communication between binding sites. The emerging picture is consistent with a binding mechanism that can be described as an extended conformational selection model.     

Identification of the 14 kDa bile acid transport protein of rat ileal cytosol as gastrotropin

The 14 kDa bile acid binding protein of rat ileal cytosol (I-BABP), previously shown to be the major intracellular transporter of bile acids in enterocytes, was purified by affinity chromatography and gel electrophoresis. Enzymatic digestion of I-BABP which had been electroblotted to nitrocellulose led to the recovery and sequence analysis of four peptides representing 47 residues of sequence (approximately 35% of the full sequence). All the peptide sequences displayed high levels of identity (greater than 60%) and homology (greater than 80%) to the sequences of porcine and canine gastrotropin. This high level of homology together with other features of I-BABP identify it as rat gastrotropin, establishing gastrotropin as the major intracellular bile acid carrier of rat enterocytes.     

FXRE can function as an LXRE in the promoter of human ileal bile acid-binding protein (I-BABP) gene

Ileal bile acid-binding protein (I-BABP) is a 14 kDa cytosolic protein which binds bile acids with a high affinity. It is thought to be implicated in the enterohepatic circulation of bile acids and, hence, in cholesterol homeostasis. Using a combination of in vivo and in vitro experiments, we have recently shown that I-BABP gene expression can be indirectly up-regulated by cholesterol through the activation of sterol-responsive element-binding protein 1c (SREBP1c) by liver X-receptor (LXR). We report here that I-BABP can be also a direct target for LXR. I-BABP regulation by LXR is maintained when the SREBP binding site is deleted in the I-BABP promoter and occurs, in the absence of conventional LXRE sequences, through an IR1 sequence previously identified as a farnesoid X-receptor-responsive element (FXRE). Electrophoretic mobility shift assays demonstrated that the LXR/RXR heterodimer specifically recognizes the FXRE. Collectively, these data strongly suggest that LXR can regulate the I-BABP gene by both direct and indirect mechanisms.     

Interaction between bile acids and staphylococcal polysaccharide: inhibition of capsule formation in encapsulated mutant strains (taurine+, taurine-) of Staphylococcus aureus

In a previous paper, we showed that bile acid derivatives inhibit capsule formation as well as taurine biosynthesis in a taurine+ (Tau+) encapsulated strain of Staphylococcus aureus. In the present study, binding of [14C]cholic acid [( 14C]CA) and [14C]taurocholic acid [( 14C]TA) to the staphylococcal polysaccharide antigen (SPA) of the capsular fraction was examined. The bile acids were found to bind with SPA via taurine of the Tau+ cells. [14C]CA bound with the SPA fraction of the Tau+ strain within 10-30 min, whereas 60-120 min was required in the binding of [14C]TA. Various bile acids competed with cholic acid binding to Tau+ cells which was shown by the inhibition of binding with cholic acid or taurocholic acid but not with glycholic acid. Binding of bile acid derivatives to a Tau- encapsulated mutant or to capsular material from this mutant was not observed.     

Use of novel cationic bile salts in cholesterol crystallization and solubilization in vitro

Unnatural bile salts have been synthesized with a cationic group at the side chain of natural bile acids. These cationic bile salts aggregate in water and aqueous salt solutions in a manner similar to their natural counterparts. The critical micellar concentrations of the cationic bile salts were measured using a fluorescence method. Cationic bile salts aggregated at a concentration lower than natural deoxycholic acid. Since dihydroxy bile salt micelles are well known for cholesterol dissolution/removal, the dissolution in the cationic micelles has been evaluated. The cationic analogs dissolve approximately 70 mg/dL of cholesterol, which is comparable to taurochenodeoxycholate micelle under identical bile salt concentrations. Cholesterol dissolution in cationic bile salt micelle enhanced upon adding various amounts of PC. Cholesterol crystallization was studied in model bile at various cationic bile salt concentrations. The addition of 5, 15 and 30 mM of the cationic bile salts attenuated the crystallization process, without influencing the crystal observation time or decreasing the final amount of crystals formed. All these effects were comparable to those observed with cholic acid. These findings suggest that cationic bile salts have physico-chemical properties analogous to those of natural anionic bile salts, and thus may have therapeutic potential.     

Synthesis and spectroscopic analysis of modified bile salts

For the study of hepatic bile acid transport in vivo, a series of modified bile salts were synthesized. The N-cholyl derivatives of L-leucine, L-alanine, D-alanine, beta-alanine, L-proline, and gamma-amino-butyric acid were prepared from cholic acid, ethyl chloroformate and the corresponding amino acid. Structural analysis of products was carried out mainly by electron impact mass spectrometry (20 eV) of the methyl ester/acetate derivatives. In all EI spectra, fragments in the lower mass region included McLafferty rearrangement ions (beta-cleavage) and product ions of gamma-cleavage in the vicinity of the amide linkage. In the upper mass region, fragmentation was characterized by consecutive eliminations of ketene and/or acetic acid from low intensity molecular ions. The purity of the products and their molecular weights were checked by a novel ionization technique in mass spectrometry, fast atom bombardment (FAB) mass spectrometry. FAB spectra were obtained from underivatized bile salts. The spectra were characterized by ions formed by attachment of a proton or an alkali ion to the bile salt to give intense M+H, M+Na, or M+K ions, which then showed little fragmentation.     

Evolution of substrate specificity for the bile salt transporter ASBT (SLC10A2)

The apical Na(+)-dependent bile salt transporter (ASBT/SLC10A2) is essential for maintaining the enterohepatic circulation of bile salts. It is not known when Slc10a2 evolved as a bile salt transporter or how it adapted to substantial changes in bile salt structure during evolution. We characterized ASBT orthologs from two primitive vertebrates, the lamprey that utilizes early 5α-bile alcohols and the skate that utilizes structurally different 5β-bile alcohols, and compared substrate specificity with ASBT from humans who utilize modern 5β-bile acids. Everted gut sacs of skate but not the more primitive lamprey transported (3)H-taurocholic acid (TCA), a modern 5β-bile acid. However, molecular cloning identified ASBT orthologs from both species. Cell-based assays using recombinant ASBT/Asbt's indicate that lamprey Asbt has high affinity for 5α-bile alcohols, low affinity for 5β-bile alcohols, and lacks affinity for TCA, whereas skate Asbt showed high affinity for 5α- and 5β-bile alcohols but low affinity for TCA. In contrast, human ASBT demonstrated high affinity for all three bile salt types. These findings suggest that ASBT evolved from the earliest vertebrates by gaining affinity for modern bile salts while retaining affinity for older bile salts. Also, our results indicate that the bile salt enterohepatic circulation is conserved throughout vertebrate evolution.     

Isolation and identification of bile salts conjugated with cysteinolic acid from bile of the red seabream, Pagrosomus major.

Bile salts present in gallbladder of wild and cultured red seabream, Pagrosomus major, a marine teleost were analyzed. The bile from wild red seabream was found to contain two previously unknown bile salts along with two known bile salts, taurocholate and taurochenodeoxycholate. Isolation of each bile salt was performed by column chromatography. Fast atom bombardment mass spectra of the unknown bile salts showed the molecular ions (M-H)- of m/z 544 and 528 which are shifted 30 mass units upfield compared to those (m/z 514 and 498) of taurocholate and taurochendeoxycholate, respectively; this is consistent with the presence of cysteinolic acid (mol wt 155) instead of taurine (mol wt 125). Enzymatic hydrolysis of the bile salts released cholic acid and chenodeoxycholic acid, respectively, and an amino acid that was identified as D-cysteinolic acid by direct comparison with an authentic sample. From these results, the bile salts in the bile of wild red seabream were identified as the conjugates of cholic acid and chenodeoxycholic acid with cysteinolic acid. 1H- and 13C-magnetic resonance spectra of the bile salts were also consistent with the proposed structure. The cysteinolic acid conjugates were found only in wild and not in cultured red seabream; this distinction seems to result from differences in dietary cysteinolic acid.     

Purification and properties of subunits of sterol ester hydrolase from rat pancreas.

A procedure has been developed for solubilizing subunits of sterol ester hydrolase (EC 3.1.1.13) from rat pancreas by treatment of a tissue homogenate with 0.5% ($\text{w}\text{v}$) digitonin. The crude, solubilized enzyme subunit was shown to have a molecular weight of approximately 70,000 by Sephadex G-200 gel filtration. This enzyme subunit has been purified 500-fold by a combination of ammonium sulfate fractionation, hydroxylapatite, and gel-filtration chromatography. The 70,000 molecular-weight subunits have been shown to aggregate in the presence of cholic acid or sodium taurocholate to a 400,000 molecular-weight form which is the active enzyme. Studies on binding of cholic acid to the subunit protein suggest that after the binding of one molecule of the bile acid, the subunit undergoes a conformational change(s) which makes additional binding sites for cholic acid available. Three types of differential inactivation studies (thermal, guanidine hydrochloride, and pH) indicated significantly greater stability of the active enzyme when compared to the subunits. These data are consistent with the tentative conclusion that a conformational change(s) accompanies the binding of the bile salt to the enzyme subunits, which results in their aggregation and enzyme activity. The theoretical and physiological significance of this interaction between the subunit protein and bile salt is discussed.     

The X-ray structure of zebrafish (Danio rerio) ileal bile acid-binding protein reveals the presence of binding sites on the surface of the protein molecule

The ileal bile acid-binding proteins (I-BABPs), also called ileal lipid-binding proteins or gastrotropins, belong to the family of the fatty acid-binding proteins and play an important role in the solubilization and transport of bile acids in the enterocyte. This article describes the expression, purification, crystallization, and three-dimensional structure determination of zebrafish (Danio rerio) I-BABP both in its apo form and bound to cholic acid. This is the first X-ray structure of an I-BABP. The structure of the apoprotein was determined to a resolution of 1.6 Å, and two different monoclinic crystal forms of the holoprotein were solved and refined to 2.2 Å resolution. Three protein molecules are present in the asymmetric unit of one of the co-crystal forms and two in the other, and therefore, the results of this study refer to observations made on five different protein molecules in the crystalline state. In every case, two cholate ligands were found bound in approximately the same position in the internal cavity of the protein molecules, but an unexpected result is the presence of clear and unambiguous electron density for several cholate molecules bound on hydrophobic patches on the surface of all the five independent protein molecules examined. Isothermal titration calorimetry was used for the thermodynamic characterization of the binding mechanism and has yielded results that are consistent with the X-ray data. Ligand binding is described in detail, and the conformational changes undergone by the protein molecule in the apo-to-holo transition are examined by superposition of the apo- and holoprotein models. The structure of the holoprotein is also compared with that of the liver BABP from the same species and those of other I-BABPs determined by NMR.     

Binding of bile acids by glutathione S-transferases from rat liver

Binding of bile acids and their sulfates and glucuronides by purified GSH S-transferases from rat liver was studied by 1-anilino-8-naphthalenesulfonate fluorescence inhibition, flow dialysis, and equilibrium dialysis. In addition, corticosterone and sulfobromophthalein (BSP) binding were studied by equilibrium and flow dialysis. Transferases YaYa and YaYc had comparable affinity for lithocholic (Kd approximately 0.2 microM), glycochenodeoxycholic (Kd approximately to 60 microM), and cholic acid (Kd approximately equal 60 microM), and BSP (Kd approximately 0.09 microM). YaYc had one and YaYa had two high affinity binding sites for these ligands. Transferases containing the Yb subunit had two binding sites for these bile acids, although binding affinity for lithocholic acid (Kd approximately 4 microM) was lower than that of transferases with Ya subunit, and binding affinities for the other bile acids were comparable to the Ya family. Sulfated bile acids were bound with higher affinity and glucuronidated bile acids with lower affinity by YaYa and YaYc than the respective parent bile acids. In the presence of GSH, binding of lithocholate by YaYc was unchanged and binding by YbYb' was inhibited. Conversely, GSH inhibited the binding of cholic acid by YaYc but had less effect on binding by YbYb'. Cholic acid did not inhibit the binding of lithocholic acid by YaYa.     

Calcium binding by monosulfate esters of taurochenodeoxycholate

The effect of sulfate esterification of the 3 alpha- or 7 alpha-hydroxyl groups of taurochenodeoxycholate on calcium binding was studied at 0.154 M NaCl in the presence and absence of phosphatidylcholine using a calcium electrode. For comparison, similar studies were made with taurochenodeoxycholate, taurodeoxycholate, and taurocholate. No high affinity calcium binding was demonstrable for any of these bile salts in pre-micellar solutions. Taurine-conjugated bile salts have greater affinity for calcium when in a micellar form. At elevated bile salt concentrations, the calcium binding of unsulfated dihydroxy taurine conjugates was similar to that of the monosulfate esters of taurochenodeoxycholate. The presence of phosphatidylcholine decreased calcium binding of the unsulfated dihydroxy bile salts and slightly increased calcium binding by taurocholate. However, the addition of phosphatidylcholine to monosulfate esters of taurochenodeoxycholate results in large increments in calcium binding. The results indicate that increased calcium binding due to the presence of phosphatidylcholine in bile salt solutions depends, in part, on the hydrophilicity of the bile salt and that the interaction of monosulfate esters of taurochenodeoxycholate with phosphatidylcholine leads to the formation of a high affinity calcium binding site.     

Hepatic overexpression of murine Abcb11 increases hepatobiliary lipid secretion and reduces hepatic steatosis

Abcb11 encodes for the liver bile salt export pump, which is rate-limiting for hepatobiliary bile salt secretion. We employed transthyretin-Abcb11 and BAC-Abcb11 transgenes to develop mice overexpressing the bile salt export pump in the liver. The mice manifest increases in bile flow and biliary secretion of bile salts, phosphatidylcholine, and cholesterol. Hepatic gene expression of cholesterol 7alpha-hydroxylase and ileal expression of the apical sodium bile salt transporter are markedly reduced, whereas gene expression of targets of the nuclear bile salt receptor FXR (ileal lipid-binding protein, short heterodimer partner (SHP) is increased. Because these changes in gene expression are associated with an increased overall hydrophobicity of the bile salt pool and a 4-fold increase of the FXR ligand taurodeoxycholate, they reflect bile salt-mediated regulation of FXR and SHP target genes. Despite the increased biliary secretion of bile salts, fecal bile salt excretion is unchanged, suggestive of an enhanced enterohepatic cycling of bile salts. Abcb11 transgenic mice fed a lithogenic (high cholesterol/fat/cholic acid) diet display markedly reduced hepatic steatosis compared with wild-type controls. We conclude that mice overexpressing Abcb11 display an increase in biliary bile salt secretion and taurodeoxycholate content, which is associated with FXR/SHP-mediated changes in hepatic and ileal gene expression. Because these mice are resistant to hepatic lipid accumulation, regulation of Abcb11 may be important for the pathogenesis and treatment of steatohepatitis.