title: Structural and molecular determinants affecting the interaction of retinol with human CRBP1.
abstract: Four cellular retinol-binding protein (CRBP) types (CRBP1,2,3,4) are encoded in the human genome. Here, we report on X-ray analyses of human apo- and holo-CRBP1, showing nearly identical structures, at variance with the results of a recent study on the same proteins containing a His-Tag, which appears to be responsible for a destabilizing effect on the apoprotein. The analysis of crystallographic B-factors for our structures indicates that the putative portal region, in particular α-helix-II, along with Arg58 and the E-F loop, is the most flexible part of both apo- and holoprotein, consistent with its role in ligand uptake and release. Fluorometric titrations of wild type and mutant forms of apo-CRBP1, coupled with X-ray analyses, provided insight into structural and molecular determinants for the interaction of retinol with CRBP1. An approximately stoichiometric binding of retinol to wild type apo-CRBP1 (Kd∼4.5nM), significantly lower binding affinity for both mutants Q108L (Kd∼65nM) and K40L (Kd∼70nM) and very low binding affinity for the double mutant Q108L/K40L (Kd∼250nM) were determined, respectively. Overall, our data indicate that the extensive apolar interactions between the ligand and hydrophobic residues lining the retinol binding cavity are sufficient to keep it in its position bound to CRBP1. However, polar interactions of the retinol hydroxyl end group with Gln108 and Lys40 play a key role to induce a high binding affinity and specificity for the interaction.
authors.affiliation: Department of Life Sciences, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy; Department of Physics and Earth Sciences, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Menozzi
authors.affiliation: Department of Biomedical Sciences, University of Padua, Via Ugo Bassi 58/B, 35131 Padua, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Vallese
authors.affiliation: Department of Physics and Earth Sciences, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy.
authors.forename: Eugenia
authors.initials: E
authors.lastname: Polverini
authors.affiliation: Department of Food Sciences, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Folli
authors.affiliation: Department of Life Sciences, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy. Electronic address: rodolfo.berni@unipr.it.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Berni
authors.affiliation: Department of Biomedical Sciences, University of Padua, Via Ugo Bassi 58/B, 35131 Padua, Italy. Electronic address: giuseppe.zanotti@unipd.it.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Zanotti
mesh_terms: D018360:Crystallography, X-Ray; D006801:Humans; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D054840:Retinol-Binding Proteins, Cellular / Q000378:metabolism*; D014801:Vitamin A / Q000378:metabolism*
chemical_list: C422994:RBP7 protein, human; D054840:Retinol-Binding Proteins, Cellular; D014801:Vitamin A
keywords: Intracellular lipid-binding proteins; Molecular dynamics simulation; Mutational analyses; Retinoid-binding proteins; Vitamin A