NMR Structural and interaction studies of bile acid binding proteinscomplexed with physiological ligands and bile acid-derived contrastagents

The research project of my PhD concerns essentially the Nuclear Magnetic Resonance (NMR) structural studies of protein-ligand complexes. Studying protein interactions at the molecular level is crucial to the understanding of many biological processes, such as human diseases and drug design. NMR spectroscopy is particularly well suited to the investigation, at the atomic level, of the interactions between proteins and other molecules in solution. NMR can be used to evaluate the structural, thermodynamic and kinetic aspects of a binding reaction, even for weak protein-ligand interactions. In this contest, during my PhD I addressed the structural investigation and the study of the molecular determinants of binding of different bile acid binding proteins (BABPs) and their native ligands or synthetic drugs. BABPs are small cytosolic proteins that display their function in the hepatocytes and enterocytes where they act as bile acids transporters participating to the enterohepatic circulation. The first part of this thesis is related to the structural determination of chicken liver-BABP (cL-BABP) in complex with a bile acid-based gadolinium(III)-chelate, a potential hepatospecific contrast agent for magnetic resonance imaging (MRI). The rationale of this study derived from a search for new hepatospecific MRI contrast agents for the discrimination and diagnosis of focal lesions or hepatic malignancies. The second part of this thesis is focused on the characterization of the interactions of chicken ileal BABP (cI-BABP) with bile acids. This study has been performed using different approaches, such as NMR spectroscopy and calorimetric measurements. The third part of the my thesis work is based on the NMR structural determination of the ternary complex between cI-BABP and two molecules of glycochenodeoxycholic acid (GCDA). Finally, a single and double mutant of cI-BABP (A101S and H99Q/A101S) were produced to investigate the molecular determinants of binding cooperativity. The obtained data allow shedding light on the chemical basis of intracellular bile acid transport. This notion may open new avenues for exploration of strategies for prevention and treatment of metabolic diseases.