Silvaroli Josie A, Arne Jason M, Chelstowska Sylwia, Kiser Philip D, Banerjee Surajit, Golczak Marcin
From the Department of Pharmacology and.
From the Department of Pharmacology and the Laboratory of Hematology and Flow Cytometry, Department of Hematology, Military Institute of Medicine, Warsaw 04-141, Poland.
J Biol Chem. 2016 Apr 15;291(16):8528-40. doi: 10.1074/jbc.M116.714535. Epub 2016 Feb 21.
Important in regulating the uptake, storage, and metabolism of retinoids, cellular retinol-binding protein 1 (CRBP1) is essential for trafficking vitamin A through the cytoplasm. However, the molecular details of ligand uptake and targeted release by CRBP1 remain unclear. Here we report the first structure of CRBP1 in a ligand-free form as well as ultra-high resolution structures of this protein bound to either all-trans-retinol or retinylamine, the latter a therapeutic retinoid that prevents light-induced retinal degeneration. Superpositioning of human apo- and holo-CRBP1 revealed major differences within segments surrounding the entrance to the retinoid-binding site. These included α-helix II and hairpin turns between β-strands βC-βD and βE-βF as well as several side chains, such as Phe-57, Tyr-60, and Ile-77, that change their orientations to accommodate the ligand. Additionally, we mapped hydrogen bond networks inside the retinoid-binding cavity and demonstrated their significance for the ligand affinity. Analyses of the crystallographic B-factors indicated several regions with higher backbone mobility in the apoprotein that became more rigid upon retinoid binding. This conformational flexibility of human apo-CRBP1 facilitates interaction with the ligands, whereas the more rigid holoprotein structure protects the labile retinoid moiety during vitamin A transport. These findings suggest a mechanism of induced fit upon ligand binding by mammalian cellular retinol-binding proteins.
细胞视黄醇结合蛋白1（CRBP1）在调节视黄醇的摄取、储存和代谢中起重要作用，对于维生素A在细胞质中的运输至关重要。然而，CRBP1摄取配体和靶向释放配体的分子细节仍不清楚。在此，我们报道了CRBP1无配体形式的首个结构以及该蛋白与全反式视黄醇或视黄胺结合的超高分辨率结构，视黄胺是一种可预防光诱导视网膜变性的治疗性类视黄醇。人源脱辅基和全蛋白形式的CRBP1叠加显示，在类视黄醇结合位点入口周围的片段内存在重大差异。这些差异包括α螺旋II以及β链βC-βD和βE-βF之间的发夹环，还有几个侧链，如Phe-57、Tyr-60和Ile-77，它们改变方向以容纳配体。此外，我们绘制了类视黄醇结合腔内的氢键网络，并证明了它们对配体亲和力的重要性。对晶体学B因子的分析表明，脱辅基蛋白中有几个区域的主链流动性较高，在结合类视黄醇后变得更加刚性。人源脱辅基CRBP1的这种构象灵活性促进了与配体的相互作用，而更刚性的全蛋白结构在维生素A运输过程中保护了不稳定的类视黄醇部分。这些发现提示了哺乳动物细胞视黄醇结合蛋白在配体结合时的诱导契合机制。
