Expression and crystallographic studies of the ligand-binding region of the human endocytic collagen receptor uPARAP
Expression and crystallographic studies of the ligand-binding region of the human endocytic collagen receptor uPARAP
复制标题
人内吞胶原蛋白受体 uPARAP 配体结合区的表达和晶体学研究
DOI：
10.1107/s2053230x15018944
复制
发表时间：
2015
影响因子：
0.9
通讯作者：
Huang Mingdong
中科院分区：
文献类型：
--
作者：
Yuan Cai;Huang Joy He;Liu Min;Huang Mingdong
尿激酶纤溶酶原激活剂受体相关蛋白（uPARAP）是一种内吞受体，可内化胶原蛋白以进行溶酶体降解，并在基质重塑中发挥重要作用。先前在巴斯德毕赤酵母中的uPARAP重组蛋白生产产生具有高度异质聚糖的蛋白质，所述高度异质聚糖易于蛋白水解降解，导致高度孪生晶体。在这项研究中，uPARAP配体结合区在稳定转染的果蝇S2昆虫细胞中表达。重组蛋白经金属亲和层析和阴离子交换层析纯化后为均一蛋白。在两种不同的pH值（5.3和7.4）下获得晶体，并分别衍射至2.44和3.13 nm分辨率。 采用分子置换结合自动构建的方法，得到uPARAP配体结合区的模型。作为甘露糖受体家族的第一个多结构域晶体结构，uPARAP配体结合区的结构表征将提供对甘露糖受体家族的pH诱导的构象重排的洞察。
Urokinase plasminogen activator receptor-associated protein (uPARAP) is an endocytic receptor that internalizes collagen for lysosomal degradation and plays an important role in matrix remodelling. Previous recombinant protein production of uPARAP in Pichia pastoris generated protein with highly heterogeneous glycans that was prone to proteolytic degradation, resulting in highly twinned crystals. In this study, the uPARAP ligand-binding region was expressed in stably transfected Drosophila S2 insect cells. The recombinant protein was homogeneous after purification by metal-affinity and anion-exchange chromatography. Crystals were obtained at two different pH values (5.3 and 7.4) and diffracted to 2.44 and 3.13 Å resolution, respectively. A model of the ligand-binding region of uPARAP was obtained by molecular replacement combined with autobuilding. As the first multidomain crystal structure of the mannose receptor family, structural characterization of the uPARAP ligand-binding region will provide insight into the pH-induced conformational rearrangements of the mannose receptor family.