Dong Hui-Jun, Tao Sheng-Mao, Li Yong-Quan, Chan Siu-Hong, Shen Xue-Ling, Wang Chun-Xia, Guan Wen-Jun
Institute of Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310027, China.
Acta Biochim Biophys Sin (Shanghai). 2006 Jul;38(7):467-76. doi: 10.1111/j.1745-7270.2006.00186.x.
Fifty-four homolog (Ffh) and FtsY are the central components of the signal recognition particle secretory pathway of bacteria. In this study, the core domain and active sites of FtsY and Ffh from Streptomyces coelicolor, which are responsible for guanosine triphosphate (GTP) hydrolysis, were identified using site-directed mutagenesis. Mutations were introduced to the conserved GXXGXGK loop of the putative GTP binding site. Mutation of the Lys residue to Gly in both FtsY and Ffh NG domains significantly decreased the GTPase activity and GTP binding affinity. Furthermore, a structural model of the ternary complex of FtsY/Ffh NG domains and the non-hydrolyzable GTP analog guanylyl 5'-(beta,gamma-methylenediphosphonate) also revealed that each Lys residue in GXXGXGK of FtsY and Ffh provides the predicted hydrogen bond required for GTP binding. However, in FtsY not in Ffh, mutation of the first Gly residue in the GXXGXGK loop disrupted the GTPase activity. In addition, protease-digesting test demonstrated that NG protein with the mutation of Lys residue was decomposed more easily. Western blot analysis suggested that in Streptomyces coelicolor, FtsY is present in the membrane fraction and Ffh in the cytosol fraction during the mid-log phase of growth. These results indicated that Lys residue in the putative GTP binding loop was the crucial residue for the GTPase activity of NG domain.
54 同源物（Ffh）和 FtsY 是细菌信号识别颗粒分泌途径的核心组成部分。在本研究中，利用定点诱变鉴定了天蓝色链霉菌中 FtsY 和 Ffh 的核心结构域及活性位点，这些结构域负责三磷酸鸟苷（GTP）水解。在假定的 GTP 结合位点的保守 GXXGXGK 环中引入突变。FtsY 和 Ffh NG 结构域中 Lys 残基突变为 Gly 显著降低了 GTP 酶活性和 GTP 结合亲和力。此外，FtsY/Ffh NG 结构域与不可水解的 GTP 类似物鸟苷 5'-（β，γ-亚甲基二膦酸酯）的三元复合物的结构模型还表明，FtsY 和 Ffh 的 GXXGXGK 中的每个 Lys 残基提供了 GTP 结合所需的预测氢键。然而，在 FtsY 而非 Ffh 中，GXXGXGK 环中第一个 Gly 残基的突变破坏了 GTP 酶活性。此外，蛋白酶消化试验表明，Lys 残基突变的 NG 蛋白更容易被分解。蛋白质印迹分析表明，在天蓝色链霉菌中，在生长对数中期，FtsY 存在于膜组分中，Ffh 存在于细胞质组分中。这些结果表明，假定的 GTP 结合环中的 Lys 残基是 NG 结构域 GTP 酶活性的关键残基。
