Probing the positioning of CRBPs upon phospholipid bilayers interaction by a suite of NMR experiments

Vitamin A must be adequately distributed within the mammalian body to produce visual chromophore in the eyes and all-trans-retinoic acid in other tissues. The cellular trafficking and metabolism of vitamin A are regulated primarily by specific high-affinity carriers called CRBP-I and CRBP-II. Both proteins deliver retinol to microsomal membrane-bound enzymes, either for esterification with fatty acids (LRAT) [1, 2] or for oxidation to retinaldehyde (RDH) [3]. 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 [3]. To address this hypothesis, we have performed a suite 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. Besides NMR, other biophysical techniques were employed to achieve a better understanding of the ongoing processes. TROSY spectra provided insights into the involved protein residues and conformational dynamics. The interaction with the liposomes differs significantly between the two homologous proteins; moreover, it depends upon the phospholipid charge and ionic strength, suggesting an electrostatic nature of the binding. The data indicate that holo CRBP-I, in contrast to CRBP-II, interacts more strongly with the anionic lipid vesicles and some conformational changes are observed in the ligand entry portal. Nevertheless, there is apparently no protein region embedded inside the bilayer, as judged by H/D exchange measurements. This study may help to understand certain aspects of the mechanisms of ligand delivery by CRBPs. References [1] J. Amengual, M. Golczak, K. Palczewski, and J.von Lintig J. Biol. Chem. 287, 24216-24227 (2012). [2] W. Jiang and J.L. Napoli Biochim. Biophys. Acta 1820, 859-869 (2012). [3] J.L. Napoli Biochim. Biophys. Acta 1821, 152-167 (2012).