Biomolecular NMR, a versatile tool for the understanding of protein science: retinoid-binding proteins as an example

Protein science stands at the heart of modern life sciences because it unravels fundamental biological mechanisms and forms the basis for rapid advances in biomedicine and biotechnology. NMR spectroscopy is uniquely suited to study various aspects of protein structure, dynamics, molecular interactions and function, because information for individual residues can be obtained; moreover, kinetic data, low-populated states and the possible formation of intermediates on the reaction pathway can be determined. The case of retinoid-binding proteins is discussed here, as an example. Vitamin A has diverse biological functions and is essential for human survival. It circulates in blood bound to serum retinol binding protein (RBP) and is transported into cells by a membrane receptor termed STRA6 (1). The cellular trafficking and metabolism of vitamin A are regulated primarily by specific high-affinity carriers called CRBP-I and CRBP-II. They represent an interesting case where structure determination as well as the study of fast dynamics (ps-ns time scale) (2) failed to elucidate the mode of retinol binding and thus to explain their diverse tissue distribution, functional roles and different ligand affinities. The highly similar structure of the apo and holo forms (a beta-barrel with two short alpha-helices, see the cartoon) exhibits a closed conformation in both proteins, that seemingly offer no access for the ligand. Given the biological relevance of retinoids, the characterization of their protein interactions and targeted release is of special interest. To tackle this challenging subject we have employed a suite of NMR techniques: 15N relaxation dispersion experiments to investigate the proteins dynamics in the slower micros-ms timescale, line-shape analysis of 15N-HSQC spectra recorded during a retinol titration to get insights into the mechanism of ligand binding and H/D exchange experiments to investigate conformational stability. The results allowed to derive a model of retinol uptake, which is different for CRBP-I and CRBP-II (3, 4); moreover, a distinct local flexibility was found to modulate their binding properties (5). The two proteins deliver retinol to microsomal membrane-bound enzymes, either for esterification with fatty acids (LRAT) (6, 7) or for oxidation to retinaldehyde (RDH) (8). Our current understanding of these processes remains incomplete, but there is evidence that the membrane microenvironment plays a role in the interactions of holo CRBPs with enzymes (8). To address this hypothesis, we have performed a series of NMR experiments with retinol-bound CRBP-I and CRBP-II in the presence of model membranes composed of either anionic or zwitterionic phospholipids, at varying protein:lipid molar ratios and ionic strength. Both homologues interact with liposomes of anionic phospholipids, but in a significantly different way (9). A conformational rearrangement of the portal region, coupled to a change in protein dynamics, are required for retinol exchange; these processes seem to be triggered by a membrane-collision. All the differences between CRBP-I and CRBP-II, when dissolved either in buffer or in the presence of biomembrane mimetic systems, may account for their distinct functional roles in the modulation of intracellular retinoid metabolism. Further experiments are in progress to better describe the ongoing processes in a biological context. (1) Kawaguchi R., Yu J., Honda J., Hu J., Whitelegge J., Ping P., Wiita P., Bok D., Sun H. (2007) Science, 315, 820-825. (2) Franzoni L., Lücke C., Pérez C., Cavazzini D., Rademacher M., Ludwig C., Spisni A., Rossi G.L., Rüterjans H. (2002) J. Biol. Chem., 277, 21983-21997. (3) Mittag T., Franzoni L., Cavazzini D., Schaffhausen B., Rossi G.L., Günther U.L. (2006) J. Am. Chem. Soc., 128, 9844-9848. (4) Franzoni L., Reed M., Cavazzini D., Rossi G.L., Günther U.L., in preparation. (5) Franzoni L., Cavazzini D., Rossi G.L., Lücke C. (2010) J. Lipid Res., 51, 1332-1343. (6) Amengual J., Golczak M., Palczewski K., von Lintig J. (2012) J. Biol. Chem., 287, 24216-24227. (7) Jiang W., Napoli J.L. (2012) Biochim. Biophys. Acta, 1820, 859-869. (8) Napoli J.L. (2012) Biochim. Biophys. Acta, 1821, 152-167. (9) Franzoni L., Baroni F., Cavazzini D., Rossi G.L., Lücke C., in preparation.