A Single Amino Acid Mutation in Zebrafish (Danio rerio) Liver Bile Acid-binding Protein Can Change the Stoichiometry of Ligand Binding

Abstract

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 threedimensional structure determination of zebrafish (Danio rerio) L-BABP to 1.5 Å 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.The liver bile acid-binding proteins, formerly called liver "basic" fatty acid-binding proteins (FABPs), 2 belong to the conserved multigene family of the fatty acid-binding proteins (1-6). Originally, the different members of this family were named according to the tissue from which they were first isolated. More recently, an alternative nomenclature has been proposed, since it is well known that different FABP types can be present in the same tissue (7). In particular, in the liver of several vertebrates, two paralogous groups of FABPs have been described: the liver FABPs and another group that was initially called the liver "basic" FABPs and is currently referred to as the liver bile acid-binding proteins (L-BABPs) (8, 9). The reason for this unusual nomenclature was that the isoelectric point of the first protein of this group that was described, chicken liver BABP, is 9.0, and although it was evident that this protein was different from the canonical liver FABP, its specific ligand was unknown (10). When it was shown that this protein could specifically bind cholic acid, a change in its designation was suggested to avoid confusion (11). The cytosolic bile acid-binding proteins mediate the intracellular movement of bile acids to the membranes across which they exit or enter the cells via membrane-bound transporters (12). The L-BABPs have been shown to be present in birds, amphibians, reptiles, and fish but not in mammals that express the very similar ileal BABPs, formerly called gastrotropins. It is suspected, although not proved, that the function that they perform ...

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“…There is, however, no similarity in the ligand-binding residues between PUR-␣ or Homer proteins and OspE, which is hardly surprising given the difference in the charge properties of the two ligands. The fatty acid-binding proteins that have a ␤-sheet structure similar to that of OspE mainly interact with the fatty acids via the residues inside the hydrophobic core of the protein (55,59,60).…”

“…A DALI (52) search showed that the globular domain of OspE resembles the SsgA-like protein (SALP) family of proteins (54), certain DNA/RNA-binding proteins (55)(56)(57)(58), some fatty acidbinding proteins (55,59,60), and the "Homer" family of proteins (61, 62) (supplemental Fig. 5).…”

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