New insights on the protein-ligand interaction differences between the two primary cellular retinol carriers

The main retinol carriers in the cytosol are the cellular retinol-binding proteins types I and II (CRBP-I, CRBP-II), which exhibit distinct tissue distributions. They play different roles in the maintenance of vitamin A homeostasis and feature a 100-fold difference in retinol affinity whose origin has not been described in detail. NMR-based hydrogen/deuterium exchange measurements show that, while retinol binding endows both proteins with a more rigid structure, many amide protons exchange much faster in CRBP-II than in CRBP-I in both apo and holo form, despite the conserved three-dimensional fold. The remarkable difference in intrinsic stability between the two homologues appears to modulate their binding properties: the stronger retinol-binder CRBP-I displays a reduced flexibility of the backbone structure with respect to CRBP-II. This difference must derive from specific evolution-based amino acid substitutions, resulting in additional stabilization of the CRBP-I scaffold: in fact, we have identified a number of potential salt-bridges on the protein surface as well as several key interactions inside the binding cavity. Furthermore, our NMR data demonstrate that helix alpha-II of the characteristic helix-turn-helix motif in the ligand portal region exists in both apo and holo CRBP-II. As a consequence, the previously proposed model of retinol binding requires revision.