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標題: Xanthomonas campestris 之Xcc6448蛋白和cGMP的共結晶結構解析與功能上的探討
Structure and function studies of protein Xcc6448 in complex with cGMP from Xanthomonas campestris
作者: 劉俊良
Liu, Jyun-Liang
關鍵字: Xcc6448;Xcc6448;GGDEF 區域蛋白;GGDEF domain proteins
出版社: 生物化學研究所
引用: Chan, C., Paul, R., Samoray, D., Amiot, N. C., Giese, B., Jenal, U. & Schirmer, T. (2004). Structural basis of activity and allosteric control of diguanylate cyclase Proc. Natl Acad. Sci. USA, 101, 17084–17089. Wassmann, P., Chan, C., Paul, R., Beck, A., Heerklotz, H., Jenal, U. & Schirmer, T. (2007). Structure of BeF3- -modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition. Structure, 15, 915–927. De, N., Pirruccello, M., Krasteva, P. V., Bae, N., Raghavan, R. V. & Sondermann, H. (2008). Phosphorylation-independent regulation of the diguanylate cyclase WspR. PLoS Biol. 6, e67 Christen, M., Christen, B., Folcher, M., Schauerte, A. & Jenal, U. (2005). Identification and characterization of a cyclic di-GMP-specific phosphodiesterase and its allosteric control by GTP. J. Biol. Chem. 280, 30829–30837. Schmidt, A. J., Ryjenkov, D. A. & Gomelsky, M. (2005). 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The cAMP receptor-like protein CLP is a novel c-di-GMP receptor linking cell-cell signaling to virulence gene expression in Xanthomonas campestris.J. Mol. Biol. 396, 646–662. Tao, F., He, Y.-W., Wu, D.-H., Swarup, S. & Zhang, L.-H. (2010). The cyclic nucleotide monophosphate domain of Xanthomonas campestris global regulator Clp defines a new class of cyclic di-GMP effectors. J. Bacteriol. 192, 1020–1029. Benach, J., Swaminathan, S. S., Tamayo, R., Handelman, S. K., Folta-Stogniew, E., Ramos, J. E., Forouhar, F., Neely, H., Seetharaman, J., Camilli, A. & Hunt, J. F. (2007). The structural basis of cyclic diguanylate signal transduction by PilZ domains. EMBO J. 26, 5153–5166. De, N., Pirruccello, M., Krasteva, P. V., Bae, N., Raghavan, R. V. & Sondermann, H. (2008). Phosphorylation-Independent Regulation of the Diguanylate Cyclase WspR. PLoS Biol. 6, e67. Wassmann, P., Chan, C., Paul, R., Beck, A., Heerklotz, H., Jenal, U. & Schirmer, T. (2007). 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Cyclic dimeric guanosine monophosphate(c-di-GMP)是細菌中的一個二級訊息分子,在細菌中扮演著一個重要的訊息傳遞者的角色,透過c-di-GMP傳遞訊息,細菌能夠快速的適應環境的變化。c-di-GMP是由具有diguanylate cyclase(DGC)酵素活性的GGDEF domain proteins合成,其酵素活性是將兩分子的GTP合成一分子的c-di-GMP。參閱目前已解析出來的全長的GGDEF domain proteins結構WspR及PleD發現,此類蛋白通常含有至少二個domains,即N端的sensorry domain和C端的GGDEF domain。N端的sensorry domain在和訊息分子結合之後,通常會改變GGDEF domain的構形,使兩個GGDEF domain聚合而形成一個雙聚體結構。在本研究中,我們透過X-ray晶體繞射技術解析出Xcc6448的cyclic nucleotide binding domain與二級訊息cGMP結合的複合體結構,其解析度為2.12 Å。在晶體結構中,一個不對稱單元包含了兩個Xcc64481~140-cGMP蛋白複合體分子。經由ITC實驗證明,Xcc64481~140和cGMP之間的結合力可達到3.5μM的程度。另一方面,我們利用pyrophosphate assay的實驗,得到初步結果顯示cGMP的結合確實能夠促進Xcc6448合成c-di-GMP的酵素活性。

Cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger that plays an important role in signal transduction in bacteria. Through this signal transduction, bacteria can adapt to fast-changing environment with the help of c-di-GMP. C-di-GMP is synthesized by GGDEF domain proteins contaning diguanylate cyclase (DGC) enzyme activity, which combines two molecules of GTP into one molecule c-di-GMP. According to the recently resolved full-length structures of GGDEF proteins from WspR and PleD, this type of protein usually comprises a sensory domain on the N-terminus and a C-terminal GGDEF domain on the C-terminus. After binding signal molecule, the C-terminal GGDEF domain will come close to form an active dimer. In this study, we study the structure and function of Xcc6448, which contains a cNMP binding domain and a GGDEF domain. We have solved the complex structure of cNMP domain with cyclic-GMP by X-ray diffraction crystallography to a resolution of 2.12 Å. Two Xcc64481~140-cGMP complex molecules are present in each asymmetric unit. ITC experiment indicate the binding strength (Kd) of cGMP with Xcc64481~140 is 3.5μM. In vitro pyrophosphate assay experiment also reveals that the binding of cGMP in the cNMP binding domain increase the DGC activity of this protein.
其他識別: U0005-1907201213215700
Appears in Collections:生物化學研究所

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