Cui Boyang, Fang Shasha, Xing Yangfei, Shen Yuequan, Yang Xue
State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, People's Republic of China.
Acta Crystallogr F Struct Biol Commun. 2015 Jul;71(Pt 7):870-5. doi: 10.1107/S2053230X15005956. Epub 2015 Jun 27.
Arabidopsis thaliana BAG5 (AtBAG5) belongs to the plant BAG (Bcl-2-associated athanogene) family that performs diverse functions ranging from growth and development to abiotic stress and senescence. BAG family members can act as nucleotide-exchange factors for heat-shock protein 70 (Hsp70) through binding of their evolutionarily conserved BAG domains to the Hsp70 ATPase domain, and thus may be involved in the regulation of chaperone-mediated protein folding in plants. AtBAG5 is distinguished from other family members by the presence of a unique IQ motif adjacent to the BAG domain; this motif is specific for calmodulin (CaM) binding, indicating a potential role in the plant calcium signalling pathway. To provide a better understanding of the IQ motif-mediated interaction between AtBAG5 and CaM, the two proteins were expressed and purified separately and then co-crystallized together. Diffraction-quality crystals of the complex were grown using the sitting-drop vapour-diffusion technique from a condition consisting of 0.1 M Tris-HCl pH 8.5, 2.5 M ammonium sulfate. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 64.56, b = 74.89, c = 117.09 Å. X-ray diffraction data were recorded to a resolution of 2.5 Å from a single crystal using synchrotron radiation. Assuming the presence of two molecules in the asymmetric unit, a Matthews coefficient of 2.44 Å(3) Da(-1) was calculated, corresponding to a solvent content of approximately 50%.
拟南芥BAG5（AtBAG5）属于植物BAG（Bcl-2相关抗凋亡基因）家族，该家族具有从生长发育到非生物胁迫和衰老等多种功能。BAG家族成员可通过其进化保守的BAG结构域与热休克蛋白70（Hsp70）的ATP酶结构域结合，充当Hsp70的核苷酸交换因子，因此可能参与植物中伴侣蛋白介导的蛋白质折叠调控。AtBAG5与其他家族成员的区别在于，其BAG结构域附近存在一个独特的IQ基序；该基序对钙调蛋白（CaM）结合具有特异性，表明其在植物钙信号通路中可能发挥作用。为了更好地理解IQ基序介导的AtBAG5与CaM之间的相互作用，分别表达并纯化了这两种蛋白质，然后将它们一起进行共结晶。使用坐滴气相扩散技术，从由0.1 M Tris-HCl pH 8.5、2.5 M硫酸铵组成的条件中生长出复合物的衍射质量晶体。这些晶体属于空间群P2(1)2(1)2(1)，晶胞参数a = 64.56、b = 74.89、c = 117.09 Å。使用同步辐射从单个晶体记录了分辨率为2.5 Å的X射线衍射数据。假设不对称单位中存在两个分子，计算出的马修斯系数为2.44 Å(3) Da(-1)，对应于约50%的溶剂含量。
