Dai Shuyan, Sun Cancan, Tan Kemin, Ye Sheng, Zhang Rongguang
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People's Republic of China.
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People's Republic of China; University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China.
Cell Calcium. 2017 Sep;66:78-89. doi: 10.1016/j.ceca.2017.05.016. Epub 2017 Jun 9.
Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca coordination. Additionally, despite lacking inter-repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.
真核细胞III型血小板反应蛋白重复序列（TT3R）是一种仅在哺乳动物血小板反应蛋白家族中发现的高效钙离子（Ca）结合基序。在原核纤维素酶Cel5G中也发现了TT3R，由于其重复序列内形成了二硫键，而非真核TT3R所具有的重复序列间二硫键，人们认为它丧失了钙离子结合能力。在本研究中，我们鉴定出大量属于几个不同蛋白家族的含原核TT3R的蛋白质，包括外膜蛋白A（OmpA），它是革兰氏阴性菌中连接外膜和周质肽聚糖层的一种重要结构蛋白。在此，我们报道了牙龈二氧化碳嗜纤维菌OmpA周质区的晶体结构，其包含一个由五个连续TT3R组成的连接区。OmpA-TT3R的结构围绕两个紧密配位的钙离子呈现出一种有序的结构，并证实了重复序列内存在异常二硫键。进一步的诱变研究表明，OmpA-TT3R的钙离子结合能力确实依赖于重复序列内二硫键的正确形成，这些二硫键有助于将一个保守的甘氨酸残基固定在其合适的位置以进行钙离子配位。此外，尽管缺乏重复序列间二硫键，但相邻OmpA-TT3R之间的界面通过疏水和保守的芳香-脯氨酸相互作用得到增强。
