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Publication Abstract from PubMed

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 alpha-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 approximately 4.5nM), significantly lower binding affinity for both mutants Q108L (Kd approximately 65nM) and K40L (Kd approximately 70nM) and very low binding affinity for the double mutant Q108L/K40L (Kd approximately 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.

Structural and molecular determinants affecting the interaction of retinol with human CRBP1.,Menozzi I, Vallese F, Polverini E, Folli C, Berni R, Zanotti G J Struct Biol. 2017 Jan 3. pii: S1047-8477(16)30267-2. doi:, 10.1016/j.jsb.2016.12.012. PMID:28057518^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.