Protein flexibility and ligand binding: dynamics of cellular retinol binding-protein types I and II

Retinoids are essential for many physiological processes like cell growth and differentiation, morphogenesis, inmunocompetence and vision1. To exert their function they have to be transported to the appropriate site, but due to their low solubility they need to be transported by a group of proteins called Retinoid Binding Proteins (RBP). This is the case for all-trans retinol, the alcohol derivative of vitamin A that is transported from the liver to the epithelial tissue by the cellular retinol binding-protein isoform I (CRBP-I) and binds to the isoform II (CRBP-II) in the small intestin epithelial. The two isoforms share the ligand-binding motif and the structural superposition of apo and holo forms reveals no significant differences between them. However, CRBP-I binds retinol with approximately 100-fold higher affinity than CRBP-II. NMR experiments indicate that amide protons exchange much faster in CRBP-II than in CRBP-I, thus isoform II should be more flexible than isoform I and that might explain the difference in affinity2,3. To tackle the problem from the computational view, we carried out a series of extended molecular dynamics simulations (3-5μs) of the two isoforms in their apo and holo states. The structural behavior of the systems is in agreement with the previous experimental results, as the key determinants of retinol affinity are related to the differential flexibility of the ligand portal site, specifically the ßE-ßF turn and helix α-II. Entropy analyses demonstrate the rigidification of the protein upon retinol binding. However, the differences in entropy found between apo and holo forms in CRBP-I are greater and more unfavorable in terms of binding than for CRBP-II. On the other hand, the different flexibility of both isoforms rises up a different hydration pattern of the binding site. Thus, CRBP-II exchanges water molecules with the bulk solvent more fluently than CRBP-I, and this process might play a significant role in the ligand binding/unbinding. Overall, the results indicate that the different affinity for retinol found in CRBP-I and CRBP-II results from a subtle interplay in the changes induced on protein dynamics and hydration by the ligand. References 1. Blomhoff. R; Blomhoff, H. K. J.Neurobiol., 66, 606-630, 2006 2. Franzoni, L.; Lücke, C.; Perez, C.; Cavazzini, D.; Rademacher, M.; Ludwig, C.; Spisni, A.; Rossi, G. L.; Ruterjans, H. J. Biol. Chem., 277, 21983-21997, 2002 3. Franzoni, L.; Cavazzini, D.; Rossi, G. L.Lücke, C. J. Lip. Res, 51, 1332-1343. . 2010