Menozzi Ilaria, Vallese Francesca, Polverini Eugenia, Folli Claudia, Berni Rodolfo, Zanotti Giuseppe
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.
Department of Biomedical Sciences, University of Padua, Via Ugo Bassi 58/B, 35131 Padua, Italy.
J Struct Biol. 2017 Mar;197(3):330-339. doi: 10.1016/j.jsb.2016.12.012. Epub 2017 Jan 3.
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.
人类基因组中编码了四种细胞视黄醇结合蛋白（CRBP）类型（CRBP1、2、3、4）。在此，我们报告了人脱辅基和全蛋白形式的CRBP1的X射线分析结果，显示其结构几乎相同，这与最近一项对含有His标签的相同蛋白质的研究结果不同，后者似乎对脱辅基蛋白具有去稳定作用。对我们结构的晶体学B因子分析表明，假定的门户区域，特别是α-螺旋-II，以及Arg58和E-F环，是脱辅基蛋白和全蛋白中最灵活的部分，这与其在配体摄取和释放中的作用一致。脱辅基CRBP1野生型和突变体形式的荧光滴定，结合X射线分析，为视黄醇与CRBP1相互作用的结构和分子决定因素提供了深入了解。分别测定了视黄醇与野生型脱辅基CRBP1的近似化学计量结合（Kd∼4.5nM）、对突变体Q108L（Kd∼65nM）和K40L（Kd∼70nM）的显著较低结合亲和力以及对双突变体Q108L/K40L（Kd∼250nM）的非常低结合亲和力。总体而言，我们的数据表明，配体与视黄醇结合腔内衬的疏水残基之间广泛的非极性相互作用足以使其保持与CRBP1结合的位置。然而，视黄醇羟基端基与Gln108和Lys40的极性相互作用在诱导高结合亲和力和相互作用特异性方面起关键作用。
