NMR Interaction studies of Human Liver Fatty Acid Binding Protein with putative ligands and associated proteins

Lipidic molecules such as fatty acids (FAs), eicosanoids and bile salts (BAs) are essential for cell survival because they serve as metabolic energy sources, substrates for membranes and signaling molecules for metabolic regulation. Due to their low solubility and in some case cytotoxicity they necessitate intracellular chaperons, which bind them, thus increasing their aqueous solubility. Fatty acid binding proteins (FABPs), belong to the Intracellular lipid binding proteins (iLBPs) family, a class of evolutionarily related small (14-15 KDa) cytoplasmic proteins, which have been proposed to be implicated in the transcellular transport of lipophilic ligands. Due to their participation in nuclear processes and lipid metabolism and homeostasis, they have recently been proposed as drug targets against the development of lipid related disorders. Among the other family members, liver fatty acid binding protein (LFABP), belonging to subfamily II of FABPs, is the most unique. Differently from the other FABPs, LFABP is able to accommodate two long chain FAs (LCFAs) molecules, but also a wide range of hydrophobic ligands, such as BAs, eicosanoids, Acyl-CoA esters and phospholipids. Due to its peculiar characteristics and the high concentration that it could reach within the hepatocytes (1-5% of total soluble proteins), human LFABP (HLFABP) has been hypothesized to be implicated in malaria parasites development, during the hepatic stage of the disease. The hepatic stage is asymptomatic and it would provide novel strategies for arresting the infection. UIS3 is a small transmembrane protein, presumably localized to the parasitophorous vacuolar membrane (PVM), specifically expressed in infective sporozoites, and essential for early-stage liver development. A yeast two-hybrid screen based on UIS3 of the rodent malaria parasite P. yoelii (Py-UIS3) identified mouse LFABP as an interacting host protein. In addition, a work of 2008 of Ashwani and collaborators reports a direct interaction between HLFABP and the soluble domain of UIS3 from Plasmodium falciparum (PfUIS3(130-229)). In order to gain binding information at an atomic level of resolution, the interaction between HLFABP and PfUIS3(130-229) was analyzed in detail exploiting Nuclear Mgnetic Resonance (NMR) spectroscopy. Furthermore, the direct binding of phospholipids and FAs to UIS3 was also analyzed by NMR. However, our data did not show any interaction of PfUIS3(130-229) with HLFABP and lipid molecules, calling for a redefinition of the current model of FABP-mediated lipid import by human malaria parasites. In the second part of this research project, we investigated in detail the interaction between HLFABP and fatty acids. For the first time NMR and MS spectroscopy were used in combination to characterize the binding between HLFABP and FAs. Samples of HLFABP in complex with palmitate (PA) or oleate (OA) were prepared in water and analyzed through Electron Spray Ionization mass spectroscopy (ESI-MS) to asses specificity, stoichiometry and relative affinity. Our data are in agreement with the presence of two distinct binding sites with different affinities for FAs. Competition experiments were also performed, titrating the protein with both PA and OA; OA and PA effectively compete for the same binding site within the protein binding pocket. Our results show that HLFABP has an higher affinity for unsaturated FAs. Successively, we exploited the power of 13C NMR titration data to investigate the interaction between HLFABP and 13C FAs and to get information about the ionization state of the bound ligands. Globally, we developed a method suitable for the study of other FABP family members, which, despite their favorable size are really challenging systems to be characterized by only a singular biophysical technique. In addition, this method could be also applied to the study of HLFABP in complex with other hydrophobic ligands. In the last part of this work we focused on the interaction between HLFABP and BAs, amphipathic molecules, which in the small intestine facilitate the absorption of dietary lipids, cholesterol, and fatsoluble vitamins. BAs undergo a recycling pathway between the intestine and the liver, called “enterohepatic circulation”, which allows the recovery of almost the 95% of these precious molecules. Since a BA carrier within the hepatocytes has not been identified yet, we explored the interaction between HLFABP and BAs using a wide range of biophysical techniques, involving NMR, florescence and mass spectroscopy. The interaction between HLFABP and glycocholic acid (GCA), the most abundant bile salt present in human liver, was extensively explored using NMR spectroscopy technique. NMR is one of the most powerful and versatile spectroscopic technique for molecular analysis, since it allows to characterize biological macromolecules and their complexes at an atomic level of resolution. In addition, NMR provides information about protein dynamics on a wide range of time scales. Dynamics can affect the rate and pathway of protein folding, as well as misfolding and aggregation, catalysis and also binding via induced fit or conformational selection. Thus, the determination of protein dynamics in solution is important for realizing the full spectrum of macromolecular functions and for predicting and engineering protein behavior. NMR titration experiments and 1H-1H homonuclear NOESY filtered experiments, performed with different labeling schemes, suggested that HLFABP is able to accommodate only one molecule of GCA. To complement NMR data, a model of the complex was obtained through a computational analysis, using the docking program HADDOCK. To better characterize the binding, 15N backbone relaxation experiments on HLFABP in its apo form and in complex with either GCA or OA were recorded and residue specific dynamics, on a time scale ranging from ps to ms, were obtained. Fast time scale dynamics are not significantly perturbed upon OA/GCA addition, while slow motions are retained or enhanced upon binding. Hydrogen/deuterium exchange and CLEANEX experiments were also performed to get information on solvent accessibility to individual sites and to detect protein dynamics occurring on a much slower time scale. An increase in protein stability upon GCA/OA binding was observed. For the first time NMR and fluorescence spectroscopy were combined on a BA pool, with different pattern of conjugation and hydroxylation. The NMR data show that HLFABP can interact with a wide range of bile salts, through a complex pathway, involving at least one activated state. In addition the hydrogen bond network was not significantly perturbed upon ligand addition, indicating that the scaffold of the protein is preformed to bind such kind of ligands. Through NMR spectroscopy, we demonstrated also that HLFABP exists as an ensemble of conformers in fast exchange on an NMR time scale. Fluorescence spectroscopy was used to calculate the affinity of HLFABP toward the different BAs employed in the study. An affinity in the µM range (spanning form 0.6-7.5µM) was obtained through DAUDA displacement assay, in close agreement with the ones calculated by NMR. The higher affinity was obtained for those BAs displaying high hydrophobic properties. Finally we analyzed both by NMR and mass spectroscopy the existence of an heterotypic complex constituted by HLFABP in complex with both GCA and FAs, which is likely the conformation assumed by the protein in vivo.