CRBPs: different mechanisms of ligand entry in spite of high structural similarity.

Four Cellular Retinol-Binding Protein (CRBP) isoforms, belonging to the intracellular lipid-binding protein (iLBP) family, have been reported in humans. Despite their low sequence identity they show a high structural conservation. CRBP 1 and 2, for which retinol is the endogenous ligand, bind it with high affinity, while CRBP3 and 4, for which the endogenous ligand is not yet characterized, show a very low affinity for retinol. One of the main differences concerning the binding site of the four isoforms is that the glutamine 108, one of the two key residues, is presents in CRBP1 and 2 while is replaced by a histidine in CRBP3 and 4. Our recent work about the characterization of the role of the key binding residues1, together with other previous studies, demonstrated that other unidentified factors, distinct from this substitution, are responsible of affinity differences between CRBPs. The different flexibility of the four isoforms could be an important feature to clarify this. To this aim, we have performed MD simulations of apo-proteins, observing different mobility in selected regions of the four proteins, in spite of their high structural conservation. Moreover, to understand the retinol up-take mechanism of the CRBP I and II, we have carried out MD simulations in the presence of the ligand. Our data suggest a different entry path for the retinol in CRBP1 and 2, even if involving the same “portal” region. We are performing the experimental characterization of mutant forms of the two CRBPs to validate the computational model. 1. Menozzi I, Vallese F, Polverini E, Folli C, Berni R, Zanotti G. Structural and molecular determinants affecting the interaction of retinol with human CRBP1. J Struct Biol. 2017;197(3). doi:10.1016/j.jsb.2016.12.012.