High structural and functional conservation but different ligand uptake: the role of the hydropathy profile of the protein surface.

Cellular Retinol-binding Proteins (CRBP) type I and II are beta-barrel proteins that show very high structural conservation in spite of a moderately low sequence identity and a different tissue distribution. These retinol carriers play role in the maintenance of vitamin A homeostasis, but exhibit a different affinity for the ligand (100 folds higher for CRBP-I). However, the binding site of the two isoforms is highly conserved. The mechanism of ligand uptake was investigated by means of molecular dynamics simulations, initially positioning the ligand outside the protein. For both CRBPs, the portal region formed by alfa helix II and the two loops between CD and EF strands is involved in the uptake, with a partial unfolding of the helix II. Nevertheless, a different distribution of polar and hydrophobic residues clusters at the surface of the two proteins, in particular at the barrel lid made by helix I and II, favored two different entrance pathways. In CRBP I, the retinol enters the binding cavity through a hydrophobic passage between alpha helix II and CD and EF loops, while in CRBP II the ligand, driven by a few polar interactions, sinks in the hydrophobic region between the two alpha helices. Then, in both cases, several polar residues interacting with OH-group, attract the retinol deeply inside the binding cavity. Therefore, even if the retinol uptake involves the same region, that covers the binding pocket and is intrinsically flexible, the ligand finds the better entrance pathway according to the hydropathy features of the protein surface.