Binding cooperativity and allosterism make liver fatty acid binding proteins ideal chaperones of lipids and lipid-functionalized drugs

Lipids are vital components of many biological processes and crucial in the pathogenesis of numerous common diseases, but the specific mechanisms coupling intracellular lipids to biological targets and signalling pathways are not well understood (1). Intracellular lipid chaperones known as fatty acid binding proteins (FABPs) coordinate lipid responses in cells and are strongly linked to metabolic and inflammatory pathways. FABPs display a wide range of sequence diversity, but share a common structural fold. Among these proteins, human liver FABP is increasingly attracting the interest due to its capability of regulating not only fatty acids but also bile acids pathways. This protein, which is the most abundant cytosolic protein in hepatocytes, is probably the most versatile chaperone in terms of its ligand repertoire. As a consequence of its abundance, it was suggested that L-FABPs may be involved not only in the transport of endogenous lipids, but also of exogenous lipophilic drugs. Our group is focussing its research on the elucidation of the complex binding mechanisms of FABPs (2). Here we present recent results on the structural and the dynamic characterization of protein-ligand adducts, obtained primarily by Nuclear Magnetic Resonance (NMR) spectroscopy. We describe measurements of translational diffusion and site-specific protein mobility that have highlighted functionally relevant protein regions. These data, performed both on L-FABP and on model proteins, have been combined with calorimetry and mass spectrometry data: the emerging picture is consistent with the occurrence of strong cooperative binding to multiple sites, originated by protein allosterism (3). Furthermore, we show very recent NMR data referred to experiments performed on FABPs in solutions containing phospholipids vesicles and in living cells. The obtained results suggest that these proteins can act as molecular switches allosterically activated by lipid molecules and/or membranes in the regulation of lipid trafficking. As an application of the acquired knowledge, we show the characterization of the interaction of FABPs with lipid-functionalized gadolinium chelates to be used as potential hepatospecific contrast agents for MRI (4). References: 1. Furuhashi M & Hotamisligil GS. Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets. Nat Rev Drug Discov. 2008;7(6): 489–503. Review. 2. Eliseo T, Ragona L, Catalano M, Assfalg M, Paci M, Zetta L, Molinari H & Cicero DO. 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. Biochemistry 2007;46(44):12557–12567. 3. Pedo` M, D’Onofrio M, Ferranti P, Molinari H & Assfalg M. Towards the elucidation of molecular determinants of cooperativity in the liver bile acid binding protein (Submitted). 4. Tomaselli S, Zanzoni S, Ragona L, Gianolio E, Aime S, Assfalg M & Molinari H. Solution structure of the supramolecular adduct between a liver cytosolic bile acid binding protein and a bile acid-based gadolinium(III)-chelate, a potential hepatospecific magnetic resonance imaging contrast agent. J Med Chem. 2008;51(21):6782–6792.