A Single Hydroxyl Group Governs Ligand Site Selectivity in Human Ileal Bile Acid Binding Protein Gregory P. Tochtrop, ²,‡,§ Gregory T. DeKoster, ‡ Douglas F. Covey, § and David P. Cistola* ,‡ Contribution from the Departments of Biochemistry & Molecular Biophysics and Molecular Biology & Pharmacology, Washington UniVersity School of Medicine, 660 South Euclid AVenue, Campus Box 8231, St. Louis, Missouri 63110 Received April 26, 2004; E-mail: cistola@cosine.wustl.edu Abstract: The recognition between proteins and their native ligands is fundamental to biological function. In vivo, human ileal bile acid binding protein (I-BABP) encounters a range of bile salts that vary in the number and position of steroidal hydroxyl groups and the presence and type of side-chain conjugation. Therefore, it is necessary to understand how chemical variability in the ligand affects the energetic and structural aspects of its recognition. Here we report studies of the binding site selectivity of I-BABP for glycocholic (GCA) and glycochenodeoxycholic (GCDA) acids using isotope-enriched bile salts along with two-dimensional heteronuclear NMR methods. When I-BABP is presented with either GCA or GCDA alone, the ligands bind to both sites. However, when presented with an equimolar mixture of the two bile salts, GCDA binds exclusively to site 1 and GCA to site 2. This remarkable selectivity is governed by the presence or absence of a single hydroxyl group at the C-12 position of the steroid tetracycle. The basis for this site selectivity appears to be energetic rather then steric. Introduction The basic processes by which proteins recognize and bind their native ligands are fundamental to the regulation of biological systems. Protein-ligand recognition is characterized by energetic, kinetic, structural, and dynamic features and includes a consideration of stoichiometry, specificity, affinity, cooperativity, and the mechanism by which the ligand is acquired and released. In the case of proteins that bind multiple heterogeneous ligands, the determination of site-specific affinity or ligand site selectivity can be a nontrivial task. Human ileal bile acid binding protein (I-BABP) recognizes a series of physiological bile salts that vary in the number and position of steroidal hydroxyl groups and the presence and type of side- chain conjugation. Therefore, one of the challenges with this system is to understand how chemical variability in the ligand affects the energetic and structural aspects of the recognition. Human I-BABP is a member of the intracellular lipid-binding protein family. These proteins are thought to facilitate the cellular trafficking and metabolic regulation of fatty acids, cholesterol, retinoids, vitamins, and bile salts. 1,2 Bile salts are steroidal surfactants that facilitate the absorption of dietary lipids, cholesterol, and fat-soluble vitamins in the lumen of the small intestine. 3,4 Bile salts are secreted into the proximal small intestine via the gall bladder and are efficiently recycled via a process termed enterohepatic circulation. Human I-BABP is thought to play a role in this recycling process via binding interactions occurring within the absorptive epithelial cells of the distal ileum. Bile salts, the major metabolites of cholesterol, consist of a 24-carbon cholanic acid scaffold as shown in Figure 1. Bile salts possess one or more R-hydroxyl groups at the C-3, C-7, and/or C-12 positions. Cholic and chenodeoxycholic acid derivatives, classified as primary bile salts, are biosynthesized in the liver in a ratio of 2:1 and constitute approximately 80% of the bile salt pool in humans. The remaining 20% are secondary bile salts that result from chemical transformations carried out by anaerobic bacteria in the small intestine. ² Present address: Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford St., Cambridge, MA 02138. ‡ Department of Biochemistry & Molecular Biophysics. § Department of Molecular Biology & Pharmacology. (1) Glatz, J. F.; van der Vusse, G. J. Prog. Lipid Res. 1996, 35, 243-282. (2) Veerkamp, J. H.; Maatman, R. G. Prog. Lipid Res. 1995, 34, 17-52. (3) Danielsson, H.; Sjovall, J. Sterols and bile acids; Elsevier: Amsterdam, 1985; Vol. 12. (4) Nair, P.; Kritchevsky, D. The bile acids; Plenum Press: New York, 1971. Figure 1. Chemical structures (showing steroid numbering) of the two most physiologically abundant bile salts. Published on Web 08/13/2004 11024 9 J. AM. CHEM. SOC. 2004, 126, 11024-11029 10.1021/ja047589c CCC: $27.50 © 2004 American Chemical Society