Crystal structure of axolotl (Ambystoma mexicanum) liver bile acid-binding protein bound to cholic and oleic acid

The family of the liver bile acid-binding proteins (L-BABPs), formerly called liver basic fatty acid-binding proteins (Lb-FABPs) shares fold and sequence similarity with the paralogous liver fatty acid-binding proteins (L-FABPs) but has a different stoichiometry and specificity of ligand binding. This article describes the first X-ray structure of a member of the L-BABP family, axolotl (Ambystoma mexicanum) L-BABP, bound to two different ligands: cholic and oleic acid. The protein binds one molecule of oleic acid in a position that is significantly different from that of either of the two molecules that bind to rat liver FABP. The stoichiometry of binding of cholate is of two ligands per protein molecule, as observed in chicken L-BABP. The cholate molecule that binds buried most deeply into the internal cavity overlaps well with the analogous bound to chicken L-BABP, whereas the second molecule, which interacts with the first only through hydrophobic contacts, is more external and exposed to the solvent.     

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.     

Crystal structure of chicken liver basic fatty acid-binding protein complexed with cholic acid

Two paralogous groups of liver fatty acid-binding proteins (FABPs) have been described: the mammalian type liver FABPs and the basic type (Lb-FABPs) characterized in several vertebrates but not in mammals. The two groups have similar sequences and share a highly conserved three-dimensional structure, but their specificity and stoichiometry of binding are different. The crystal structure of chicken Lb-FABP complexed with cholic acid and that of the apoprotein refined to 2.0 A resolution are presented in this paper. The two forms of the protein crystallize in different space groups, and significant changes are observed between the two conformations. The holoprotein binds two molecules of cholate in the interior cavity, and the contacts observed between the two ligands can help to explain the reason for this stoichiometry of binding. Most of the amino acids involved in ligand binding are conserved in other members of the Lb-FABP family. Since the amino acid sequence of the Lb-FABPs is more similar to that of the bile acid-binding proteins than to that of the L-FABPs, the possibility that the Lb-FABPs might be more appropriately called liver bile acid-binding proteins (L-BABPs) is suggested.     

Regulation of the ileal bile acid-binding protein gene: An approach to determine its physiological function(s)

Ileal bile acid-binding protein (I-BABP) is a soluble bile acids (BA) carrier protein which belongs to the fatty acid-binding protein (FABP) family. In the gut, its expression is strictly restricted to the ileum, where it is thought to be involved in the active BA reabsorption. Therefore, I-BABP gene expression levels might be rate limiting for the BA enterohepatic circulation, and hence, might be crucial for cholesterol (CS) homeostasis. Indeed, BA not reclaimed by intestinal absorption constitute the main way to eliminate a CS excess. However, such a function is not yet established. Because generally rate limiting genes are tightly controlled, we have undertaken the study of the I-BABP gene regulation. It was found that both BA and CS, probably via oxysterols, are able to up-regulate the trancription rate of I-BABP gene. The fact that intracellular sterol sensors (FXR, LXR and SREBP1c) are involved in the control of I-BABP gene expression strongly suggest a crucial role for I-BABP in the ileum.     

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.     

Steroid ring hydroxylation patterns govern cooperativity in human bile acid binding protein

Human ileal bile acid binding protein (I-BABP) is a member of the intracellular lipid binding protein family. This protein is thought to function in the transcellular transport and enterohepatic circulation of bile salts. Human I-BABP binds two molecules of glycocholate, the physiologically most abundant bile salt, with modest intrinsic affinity but a remarkably high degree of positive cooperativity. Here we report a calorimetric analysis for the binding of a broad panel of bile salts to human I-BABP. The interaction of I-BABP with nine physiologically relevant derivatives of cholic acid, chenodeoxycholic acid, and deoxycholic acid in their conjugated (glycine and taurine) and unconjugated forms was monitored by isothermal titration calorimetry. All bile salts bound to I-BABP with a 2:1 stoichiometry and similar overall affinity, but the derivatives of cholic acid displayed much higher Hill coefficients, a measure of macroscopic positive cooperativity. To test whether the cooperativity was dependent on individual structural features of the bile salt side chain, a series of side-chain-extended bile salts that lacked a hydrogen bond donor or acceptor at C-24 were chemically synthesized. These synthetic variants exhibited the same energetic and cooperativity profile as the naturally occurring bile salts. Our findings indicate that cooperativity in bile salt-I-BABP recognition is governed by the pattern of steroid B- and C-ring hydroxylation and not the presence or type of side-chain conjugation.     

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.     

Lupinus luteus pathogenesis-related protein as a reservoir for cytokinin

Plant pathogenesis-related (PR) proteins of class 10 (PR-10) are small and cytosolic. The main feature of their three-dimensional structure is a large cavity between a seven-stranded antiparallel β-sheet and a long C-terminal α-helix. Although PR-10 proteins are abundant in plants, their physiological role remains unknown. Recent data have indicated ligand binding as their possible biological function. The article describes the structure of a complex between a classic PR-10 protein (yellow lupine LlPR-10.2B) and the plant hormone, trans-zeatin. Previously, trans-zeatin binding has been reported in a structurally related cytokinin-specific binding protein, which has a distant sequence relation with classic PR-10 proteins. In the present 1.35 Å resolution crystallographic model, three perfectly ordered zeatin molecules are found in the binding cavity of the protein. The fact that three zeatin molecules are bound by the protein when only a fourfold molar excess of the ligand was used indicates an unusual type of affinity for this ligand and suggests that LlPR-10.2B, and perhaps other PR-10 proteins as well, acts as a reservoir of cytokinin molecules in the aqueous environment of the cell.     

Crystal structure of the peptidoglycan recognition protein at 1.8 A resolution reveals dual strategy to combat infection through two independent functional homodimers

The mammalian peptidoglycan recognition protein-S (PGRP-S) binds to peptidoglycans (PGNs), which are essential components of the cell wall of bacteria. The protein was isolated from the samples of milk obtained from camels with mastitis and purified to homogeneity and crystallized. The crystals belong to orthorhombic space group I222 with a = 87.0 Å, b = 101.7 Å and c = 162.3 Å having four crystallographically independent molecules in the asymmetric unit. The structure has been determined using X-ray crystallographic data and refined to 1.8 Å resolution. Overall, the structures of all the four crystallographically independent molecules are identical. The folding of PGRP-S consists of a central β-sheet with five β-strands, four parallel and one antiparallel, and three α-helices. This protein fold provides two functional sites. The first of these is the PGN-binding site, located on the groove that opens on the surface in the direction opposite to the location of the N terminus. The second site is implicated to be involved in the binding of non-PGN molecules, it also includes putative N-terminal segment residues (1-31) and helix α2 in the extended binding. The structure reveals a novel arrangement of PGRP-S molecules in which two pairs of molecules associate to form two independent dimers. The first dimer is formed by two molecules with N-terminal segments at the interface in which non-PGN binding sites are buried completely, whereas the PGN-binding sites of two participating molecules are fully exposed at the opposite ends of the dimer. In the second dimer, PGN-binding sites are buried at the interface while non-PGN binding sites are fully exposed at the opposite ends of the dimer. This form of dimeric arrangement is unique and seems to be aimed at enhancing the capability of the protein against specific invading bacteria. This mode of functional dimerization enhances efficiency and specificity, and is observed for the first time in the family of PGRP molecules.     

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.     

A single amino acid mutation in zebrafish (Danio rerio) liver bile acid-binding protein can change the stoichiometry of ligand binding

In all of the liver bile acid-binding proteins (L-BABPs) studied so far, it has been found that the stoichiometry of binding is of two cholate molecules per internal binding site. In this paper, we describe the expression, purification, crystallization, and three-dimensional structure determination of zebrafish (Danio rerio) L-BABP to 1.5A resolution, which is currently the highest available for a protein of this family. Since we have found that in zebrafish, the stoichiometry of binding in the protein cavity is of only one cholate molecule per wild type L-BABP, we examined the role of two crucial amino acids present in the binding site. Using site-directed mutagenesis, we have prepared, crystallized, and determined the three-dimensional structure of co-crystals of two mutants. The mutant G55R has the same stoichiometry of binding as the wild type protein, whereas the C91T mutant changes the stoichiometry of binding from one to two ligand molecules in the cavity and therefore appears to be more similar to the other members of the L-BABP family. Based on the presence or absence of a single disulfide bridge, it can be postulated that fish should bind a single cholate molecule, whereas amphibians and higher vertebrates should bind two. Isothermal titration calorimetry has also revealed the presence in the wild type protein and the G55R mutant of an additional binding site, different from the first and probably located on the surface of the molecule.     

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.     

Three-dimensional structure of recombinant human muscle fatty acid-binding protein.

The three-dimensional structure of recombinant human muscle fatty acid-binding protein with a bound fatty acid has been solved and refined with x-ray diffraction data to 2.1 A resolution. The refined model has a crystallographic R factor of 19.5% for data between 9.0 and 2.1 A (7243 unique reflections) and root-mean-square deviations in bond length and bond angle of 0.013 A and 2.7 degrees. The protein contains 10 antiparallel beta-strands and two short alpha-helices which are arranged into two approximately orthogonal beta-sheets. Difference electron density maps and a multiple isomorphous derivative electron density map showed the presence of a single bound molecule of a long chain fatty acid within the interior core of the protein. The hydrocarbon tail of the fatty acid was found to be in a "U-shaped" conformation. Seven ordered water molecules were also identified within the interior of the protein in a pocket on the pseudo-si face of the fatty acid's bent hydrocarbon tail. The methylene tail of the fatty acid forms van der Waals interactions with atoms from 13 residues and three ordered waters. The carboxylate of the fatty acid is located in the interior of the protein where it forms hydrogen bonds with the side chains of Tyr128 and Arg126 and two ordered water molecules. A comparison of the three-dimensional structure of human muscle fatty acid-binding protein and rat intestinal fatty acid-binding protein shows strong similarity. Both proteins bind a single fatty acid within their interior cores, but the bound fatty acids are very different in their conformations and interactions. These findings suggest that the intestinal and muscle fatty acid-binding proteins have evolved distinct binding sites in order to satisfy different requirements within the tissues where they are expressed.     

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.     

Crystal structure of a glutamate/aspartate binding protein complexed with a glutamate molecule: structural basis of ligand specificity at atomic resolution

The crystal structure of a periplasmic l-aspartate/l-glutamate binding protein (DEBP) from Shigella flexneri complexed with an l-glutamate molecule has been determined and refined to an atomic resolution of 1.0 Å. There are two DEBP molecules in the asymmetric unit. The refined model contains 4462 non-hydrogen protein atoms, 730 water molecules, 2 bound glutamate molecules, and 2 Tris molecules from the buffer used in crystallization. The final R_cryst and R_free factors are 13.61% and 16.89%, respectively. The structure has root-mean-square deviations of 0.016 Å from standard bond lengths and 2.35° from standard bond angles.The DEBP molecule is composed of two similarly folded domains separated by the ligand binding region. Both domains contain a central five-stranded β-sheet that is surrounded by several α-helices. The two domains are linked by two antiparallel β-strands. The overall shape of DEBP is that of an ellipsoid approximately 55 Å × 45 Å × 40 Å in size.The binding of ligand to DEBP is achieved mostly through hydrogen bonds between the glutamate and side-chain and main-chain groups of DEBP. Side chains of residues Arg24, Ser72, Arg75, Ser90, and His164 anchor the deprotonated γ-carboxylate group of the glutamate with six hydrogen bonds. Side chains of Arg75 and Arg90 form salt bridges with the deprotonated α-carboxylate group, while the main-chain amide groups of Thr92 and Thr140 form hydrogen bonds with the same group. The positively charged α-amino group of the l-glutamate forms salt bridge interaction with the side-chain carboxylate group of Asp182 and hydrogen bond interaction with main-chain carbonyl oxygen of Ser90. In addition to these hydrogen bond and electrostatic interactions, other interactions may also play important roles. For example, the two methylene groups from the glutamate form van der Waals interactions with hydrophobic side chains of DEBP.Comparisons with several other periplasmic amino acid binding proteins indicate that DEBP residues involved in the binding of α-amino and α-carboxylate groups of the ligand and the pattern of hydrogen bond formation between these groups are very well conserved, but the binding pocket around the ligand side chain is not, leading to the specificity of DEBP. We have identified structural features of DEBP that determine its ability of binding glutamate and aspartate, two molecules with different sizes, but discriminating against very similar glutamine and asparagine molecules.     

Conformational Changes and Ligand Recognition of Escherichia colid-Xylose Binding Protein Revealed

ATP binding cassette transport systems account for most import of necessary nutrients in bacteria. The periplasmic binding component (or an equivalent membrane-anchored protein) is critical to recognizing cognate ligand and directing it to the appropriate membrane permease. Here we report the X-ray structures of d-xylose binding protein from Escherichia coli in ligand-free open form, ligand-bound open form, and ligand-bound closed form at 2.15 Å, 2.2 Å, and 2.2 Å resolutions, respectively. The ligand-bound open form is the first such structure to be reported at high resolution; the combination of the three different forms from the same protein furthermore gives unprecedented details concerning the conformational changes involved in binding protein function. As is typical of the structural family, the protein has two similar globular domains, which are connected by a three-stranded hinge region. The open liganded structure shows that xylose binds first to the C-terminal domain, with only very small conformational changes resulting. After a 34° closing motion, additional interactions are formed with the N-terminal domain; changes in this domain are larger and serve to make the structure more ordered near the ligand. An analysis of the interactions suggests why xylose is the preferred ligand. Furthermore, a comparison with the most closely related proteins in the structural family shows that the conformational changes are distinct in each type of binding protein, which may have implications for how the individual proteins act in concert with their respective membrane permeases.     

Cholesterol-lowering effect of rice bran protein containing bile acid-binding proteins

Dietary plant protein is well known to reduce serum cholesterol levels. Rice bran is a by-product of rice milling and is a good source of protein. The present study examined whether feeding rats a high-cholesterol diet containing 10% rice bran protein (RBP) for 10 d affected cholesterol metabolism. Rats fed dietary RBP had lower serum total cholesterol levels and increased excretion of fecal steroids, such as cholesterol and bile acids, than those fed dietary casein. In vitro assays showed that RBP strongly bound to taurocholate, and inhibited the micellar solubility of cholesterol, compared with casein. Moreover, the bile acid-binding proteins of the RBP were eluted by a chromatographic column conjugated with cholic acid, and one of them was identified as hypothetical protein OsJ_13801 (NCBI accession No. EAZ29742) using MALDI-TOF mass spectrometry analysis. These results suggest that the hypocholesterolemic action of the RBP may be caused by the bile acid-binding proteins.     

Probing internal water molecules in proteins using two-dimensional 19F-1H NMR

Summary A simple approach for detecting internal water molecules in proteins in solution is described. This approach combines ¹⁹F-detected heteronuclear Overhauser and exchange spectroscopy (HOESY) with site-specific ¹⁹F substitution. The model system employed was intestinal fatty acid-binding protein complexed with [2-mono-¹⁹F]-palmitate. An intense cross peak was observed between the fluorine and a buried water molecule, as defined in the 1.98 Å crystal structure of the complex. From HOESY spectra, the fluorine-water distance was estimated to be 2.1 Å, in agreement with the crystal structure. This approach may be applicable to macromolecules that are too large for ¹H-detected NMR methods.