Structural and Dynamic Determinants of Ligand Binding in the Ternary Complex of Chicken Liver Bile Acid Binding Protein with Two Bile Salts Revealed by NMR^,

Abstract

Bile acids are physiological detergents facilitating absorption, transport, and distribution of lipid-soluble vitamins and dietary fats;they also play a role as signaling molecules that activate nuclear receptors and regulate cholesterol metabolism. Bile acid circulation is mediated by bile acid binding proteins (BABPs), and a detailed structural study of the complex of BABPs with bile salts has become a key issue for the complete understanding of the role of these proteins and their involvement in cholesterol homeostasis. The solution structure here reported describes, at variance with previously determined singly ligated structures, a BABP in a ternary complex with two bile acid molecules, obtained by employing a variety of NMR experiments. Exchange processes between the two bound chenodeoxycholate molecules as well as the more superficial ligand and the free pool have been detected through ROESY and diffusion experiments. Significant backbone flexibility has been observed in regions located at the protein open end, facilitating bile salts exchange. A detailed description of the protonation states and tautomeric forms of histidines strongly supports the view that histidine protonation modulates backbone flexibility and regulates ligand binding. This structure opens the way to targeted site-directed mutagenesis and interaction studies to investigate both binding and nuclear localization mechanisms.

Structural determinants of ligand binding in the ternary complex of human ileal bile acid binding protein with glycocholate and glycochenodeoxycholate obtained from solution NMR

“…2A (data not shown). Analysis of the NOE data with various combinations of bile salt 13 C/ 15 N labelling reported spatial proximities between bound bile salts and the following residues: F2, F6, M8, V40, Y53, S101, L108 (GCA), I23, F47, F63, T73, M74, G75, V83, L90 (GCDA) and W49 (GCA and GCDA). The assignment of the specific NOEs detected is listed in Table S1.…”

“…The expression of uniformly 13 C-, 15 N-, and 13 C/ 15 Nenriched human I-BABP followed a two-stage strategy designed to achieve an optimal balance between cell growth and isotope utilization. Specifically, the Escherichia coli strain MG1655, transformed with the pMON5840-hIBABP construct, was incubated overnight (37°C, 250 rpm) in 80 mL non-isotope-enriched minimal medium ( …”

“…The synthesis of [23, C 2 ]-labelled GCA and GCDA was achieved by a steroidal side chain degradation and isotopic regeneration strategy [39]. The synthesis of [3,[4][5][6][7][8][9][10][11][12][13] C 2 ]-labelled bile salts is detailed elsewhere [40]. Isotopically enriched and unenriched bile salts were dissolved in tetrahydrofuran.…”

Structural Requirements for Cooperativity in Ileal Bile Acid-binding Proteins

“…Recombinant unlabeled (u-) and 15 N, 13 C-labeled wild type and mutant I-BABPs were expressed as described previously (19 13 C-filtered, F 2 -13 C-separated, F 3 -13 C-edited NOESY-HSQC spectra (optimized for either aliphatic or aromatic residues) were recorded in D 2 O (mixing time 120 ms) for intermolecular NOE detection. The first transients of F 1 -13 C-edited, F 3 -13 C-filtered three-dimensional HMQC-NOESY experiments with mixing times ranging from 0 to 200 ms were acquired as described previously (12). 15 N-Enriched-ligand/protein ratios of 0.3, 1, 1.2, 1.5, 2, 2.2, and 4 were employed in HSQC titration experiments on 0.4 mM protein samples (either wild type or mutants).…”

“…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). One of the most intriguing features described for some members of the family relates to binding cooperativity, i.e.…”