The complex process of vitamin A uptake: a first insight by NMR and other biophysical techniques

Vitamin A is essential for diverse aspects of life, ranging from embryogenesis to the proper functioning of most adult organs. It circulates in blood bound to serum retinol binding protein (RBP) and is transported into cells by a multitransmembrane receptor termed stimulated by retinoic acid 6 (STRA6)(1). A STRA6-mediated release of retinol from holo-RBP to target organs through a new mechanism has an evolutionary advantage that prevents a possible toxicity deriving from an excessive accumulation of free vitamin A (2). There is also evidence that a specific binding site for the apo-form of the cellular carriers (CRBP) might exist on the cytoplasmic side of the membrane (3). To gain a first insight into this complex process, we have investigated the effects of biomembrane mimetic systems on CRBP-I and CRBP-II, by means of NMR, Circular Dichroism and Fluorescence measurements. The interactions of the two homologous proteins with model membranes were studied by recording 15N-HSQC and 15N-TROSY spectra at different molar ratios. Chemical shifts perturbations and line shape analysis provided insights into the interacting residues and proteins conformational dynamics. As the signals were broadened beyond detection at latest steps of the titration, the NMR data have been complemented by CD and Fuorescence measurements. The results revealed striking differences between the two homologs (4), despite having nearly identical backbone structures (a beta-barrel with two short alpha-helices) and identical retinol-binding motifs. Apo-CRBP-I interacts with model membranes and very likely the helical domain participates in the formation of a protein-membrane “collisional complex” that results in structural changes of the retinol entry site and in a slower protein tumbling. In contrast, NMR data reveal a higher structural flexibility of apo-CRBP-II that allows the existence of more than one conformation. Line shapes analysis performed in the course of the titrations indicated that the protein conformational dispersion is reduced to one preferred state in the presence of phospholipids bilayers. CD spectra showed that the overall structural integrity of the protein is not affected by the presence of the liposomes. These new evidences complement our earlier results, which had suggested that the two primary cellular retinol carriers exhibit different mechanisms of ligand uptake (5, 6); combined with their distinct tissue distribution this may imply different roles in an intracellular context. References: 1. Kawaguchi R., Yu J., Honda J., Hu J., Whitelegge J., Ping P., Wiita P., Bok D. and H. Sun Science 315, 820-825 (2007). 2. Kawaguchi R., Yu J., Ter-Stepanian M., Zhong M., Cheng G., Yuan Q., Jin M., Travis G.H., Ong D. and Sun H. ACS Chemical Biology 6, 1041-1051 (2011). 3. Redondo C., Vouropoulou M., Evans J. and Findlay J.B.C. The FASEB J. 22, 1043-1054 (2008). 4. Franzoni L., Baroni F., Cavazzini D., Rossi G.L. and Lücke C., in preparation. 5. Mittag T., Franzoni L., Cavazzini D., Schaffhausen B., Rossi G.L. and Günther U.L. J. Am. Chem. Soc. 128, 9844-9848 (2006). 6. Franzoni L., Cavazzini D., Rossi G.L. and Lücke C. J. Lipid Res. 51, 1332-1343 (2010). Acknowledgements: the EU-NMR infrastructure HWB●NMR @ Birmingham (UK) is acknowledged for providing access to instrumentation.