NMR study of chicken Liver Bile Acid Binding Protein: interaction and dynamics.

The aim of this thesis is to understand the role played by a naturally occurring disulphide bridge on the bile acid (BA) binding and functional properties of cytosolic Liver Bile Acid Binding Protein (L-BABP). Bile acids circulate between liver and intestine through a mechanism defined as “enterohepatic circulation”, which is a tightly regulated process, particularly by BAs themselves. Indeed BAs are able to influence the expression of numerous genes involved in their synthesis and transport by binding to the primary intracellular nuclear bile acid receptor, farnesoid X receptor (FXR). Understanding the mechanism regulating the interactions of intracellular carriers with bile acid is a key step to provide a model for the transfer of BAs from cytoplasm to the nucleus and can be used to inspire design of therapeutic agents in the treatment of metabolic disorders, such as obesity, type 2 diabetes, hyperlipidaemia and atherosclerosis. To achieve a detailed molecular and dynamical description of the binding mechanism driving to the formation of the ternary complex of L-BABPs with two BA molecules, spectroscopic methods together with kinetic and thermodynamic analysis have been applied and implemented. In particular structural, dynamical and interaction properties of two forms of chicken L-BABP (cL-BABP), differing by the presence/absence of a naturally occurring disulphide bridge, have been investigated through nuclear magnetic resonance (NMR) approaches. The study of protein-ligand interactions by NMR was performed analysing complexes where, alternatively, either the protein or the ligand were isotopically labelled. 15N enriched glycocholic (GCA) and glycochenodeoxycholic acid (GCDA), two of the most important members of bile salts pool, were employed for protein titrations and their resonances followed through the acquisition and analysis of several NMR experiments (HSQC, DOSY). The obtained results shed light on binding stoichiometry and ligand exchange phenomena but were not sufficient to derive detailed information on affinity, cooperativity and binding mechanism. Thus NMR lineshape analysis as a function of ligand concentration was chosen as an appropriate tool to investigate the complex interaction mechanism within the cL-BABP/BA system. In this line, new NMR approaches have been recently described which allow a reliable and sensitive investigation of ligand binding events occurring on microsecond to millisecond (μs-ms) time scales using lineshape and relaxation dispersion experiments[1]. Particularly, the combination of these NMR methods can be useful in the study of complex multi-step mechanisms, allowing the correlation between protein dynamics and function[2]. 15N relaxation studies, performed on the apo-protein, revealed the presence of slow motions occurring on the microseconds-milliseconds timescale. The central question to be addressed is here whether these motions are essential for ligand uptake, how they can eventually lead to conformations competent for binding and how they are influenced by the presence of the disulfide bridge. The analysis of titration experiments of 15N labelled protein with unlabelled GCDA through lineshape analysis and relaxation dispersion allowed to define a multi-step binding mechanism for bile salt binding to liver BABPs and to provide an estimate of the kinetics involved.