Liver bile acid intracellular transporter: NMR studies of unfolding, cooperative binding and interaction with membrane mimetic.

Bile acids are cholesterol derived molecules which, together with phospholipids, cholesterol and bilirubin, represent the principal constituents of bile. Bile acids are detergent molecules which, in the intestinal tract, help the emulsification of fat, thus improving its absorption by the intestinal epithelia. Recently another important bile acid function has emerged linked to their role in many signalling processes. Some bile acid receptors have been indeed identified and several metabolic disorders, related to impaired bile acid dependent signalling, have been reported. Bile acids undergo, in the body, a recycling pathway between the intestine and the liver, called “enterohepatic circulation”, which assure the recovery of these molecules and their subsequent reutilization. Bile acids transport is achieved, in the enterocytes and in the hepatocytes, mainly through a protein mediated mechanism that involves transmembrane and intracellular transporters. In this study the intracellular bile acid transporter from chicken liver (cL-BABP) has been characterized through NMR spectroscopy. cL-BABP is a soluble protein of about 14 KDa with the three-dimensional structure represented by a 10 stranded beta barrel with a helix-turn-helix motive that covers the open end of the barrel. In the first part of the project the stability and unfolding mechanism of cL-BABP was investigated. The structural stability was analyzed through hydrogen-deuterium (HX) exchange experiments and the ΔGop was estimated. The protein unfolding mechanism was also investigated in a residue specific manner through urea titration NMR experiments and the results compared with the data available for other members of the same protein family. In a second part of the project a cL-BABP mutant (H98Q), in which the histidine in position 98 was substituted with a glutamine, was produced and analyzed to investigate the role of such residue in triggering the allosteric binding mechanism. The overall structure of the protein was substantially unaffected by the mutation, in spite of a slightly reduced stability. The analysis of the chemical shift changes between the WT and the mutant revealed the presence of long range effects in the 55-59 segment of the protein which makes contacts with one ligand. The analysis of the binding mode, investigated both through NMR and mass spectrometry (MS), indicated some disruption of binding cooperativity as evidenced by the reduced binding affinity for one ligand. The loss of affinity for one ligand was also confirmed by the comparison of the ligand diffusion coefficients (D) in the complexes of WT and H98Q. The protein mobility was also investigated and the NMR relaxation parameters, T1 and T2, were measured. The main effect of the mutation resulted in the quenching of the slow motions in the C-terminal region of the WT protein. The last part of the project was focused on the NMR characterization of the interactions of cL-BABP with anionic vesicles in the presence of the physiological ligand, i.e. glycochenodeoxycholic acid (GCDA). Initially the association with anionic vesicles, i.e., 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) liposomes, was analyzed in the absence of bile acids and the electrostatic nature of the interaction was confirmed. Then the ternary complex cL-BABP/DMPG/GCDA was studied. The analysis of several NMR titration experiments clearly indicated that cL-BABP associates with DMPG liposomes in the apo form, while such interaction is weakened in the holo form. Indeed the addition of the physiological ligand to the protein-liposome mixture modulated this interaction, shifting the equilibrium towards the free folded holo protein. Thus membrane and ligands were shown to establish competing binding equilibria for the interaction with cL-BABP. These results supported a mechanism of ligand binding and release controlled by the onset of a bile salt concentration gradient within the polarized hepatic cell. The data obtained for the H98Q mutant allowed to clarify the key role of His98 in the modulation of the mobility observed in cL-BABP and its importance for an efficient cooperative bile acids binding. Finally, on the basis of the results of the interaction with the lisposomes, it has been proposed model for the intracellular bile acids transport in the hepatocytes that takes into account the intracellular bile acid concentration gradient.