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標題: Xanthomonas campestris 之FimXEAL-c-di-GMP 和 FimXEAL-c-di-GMP-PilZ 複合體的製備與功能分析
Preparation and functional studies of FimXEAL-c-di-GMP and FimXEAL-c-di-GMP-PilZ complexes from Xanthomonas campestris
作者: 廖怡婷
Liao, Yi-Ting
關鍵字: 複合體交互作用;FimX
出版社: 生物化學研究所
引用: Alm, R. A., Bodero, A. J., Free, P. D., and Mattick, J. S. (1996). Identification of a novel gene, pilZ, essential for type 4 fimbrial biogenesis in Pseudomonas aeruginosa. J Bacteriol 178, 46-53. Amikam, D., and Galperin, M. Y. (2006). PilZ domain is part of the bacterial c-di-GMP binding protein. Bioinformatics 22, 3-6. Benach, J., Swaminathan, S. S., Ramayo, R., Handelman, S. K., Folta-Stogniew, E., Ramos, J. E., Forouhar, F., Neely, H., Seetharaman, J., Camilli, A., and Hunt, J. F. (2007). The structural basis of cyclic diguanylate signal transduction by PilZ domains. EMBO J 26, 5153-5166. Boehm, A., Kaiser, M., Li, H., Spangler, C., Kasper, C. A., Ackermann, M., Kaever, V., Sourjik, V., Roth, V., and Jenal, U. (2010). Second messenger-mediated adjustment of bacterial swimming velocity. Cell 141, 107-116. Burdette, D., Monroe, K., Sotelo-Troha, K., Iwig, J., Eckert, B., Hyodo, M., Hayakawa, Y., and Vance, R. (2011). STING is a direct innate immune sensor of cyclic di-GMP. Nature 478, 515-518. Chan, C., Paul, R., Samoray, D., Amiot, N. C., Giese, B., Jenal, U., and Schirmer, T. (2004). Structural basis of activity and allosteric control of diguanylate cyclase. Proc Natl Acad Sci USA 101, 17084-17089. Chin, K.-H., Lee, Y.-C., Tu, Z.-L., Chen, C.-H., Tseng, Y.-H., Yang, J.-M., Ryan, R. P., McCarthy, Y., Dow, J. M., Wang, A. H.-J., and Chou, S.-H. (2010). The c-AMP 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. de La Fortelle, E., and Bricogne, G. (1997). Maximum-Likelihood Heavy-Atom Parameter Refinement for Multiple Isomorphous Replacement and Multiwavelength Anomalous Diffraction Methods. Method Enzymol 276, 472-494. De, N., Pirruccello, M., Krasteva, P. V., Bae, N., Raghavan, R. V., and Sondermann, H. (2008). Phosphorylation-independent regulation of the diguanylate cyclase WspR. PLoS Biology 6, e67. Fang, X., and Gomelsky, M. (2010). A post-translational, c-di-GMP-dependent mechanism regulating flagellar motility. Mol Microbiol 76, 1295-1305. Guzzo, C. R., Salinas, R. K., Andrade, M. O., and Farah, C. S. (2009). PilZ Protein Structure and Interactions with PilB and the FimX EAL Domain: Implications for Control of Type IV Pilus Biogenesis. J Mol Biol 393, 846-866. Habazettl, J., Allan, M. G., Jenal, U., and Grzesiek, S. (2011). Solution structure of the PilZ domain protein PA4608 complex with cyclic di-GMP identifies change clustering as molecular readout. J Biol Chem 286, 14304-14314. Hengge, R. (2009). Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol 7, 263-273. Hickman, J. W., and Harwood, C. S. (2008). Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol Microbiol 69, 376-389. Huang, B., Whitchurch, C. B., and Mattick, J. S. (2003). FimX, a multidomain protein connecting environmental signals to twitching motility in Pseudomonas aeruginosa. J Bacteriol 185, 7068-7076. Jenal, U., and Malone, J. (2006). Mechanisms of cyclic-di-GMP signaling in bacteria. Annu Rev Genet 40, 385-407. Kazmierczak, B., Lebron, M. B., and Murray, T. S. (2006). Analysis of FimX, a phosphodiesterase that governs twitching motilioty in Pseudomonas aeruginosa. Mol Microbiol 60, 1026-1043. Klausen, M., Aaes-Jorgensen, A., Molin, S., and Tolker-Nielsen, T. (2003a). Involvment of bacterial migration in the development of complex multicellular structures in Pseudomonas aeruginosa biofilms. Mol Microbiol 50, 61-68. Klausen, M., Heydorn, A., Ragas, P., Lambertsen, L., Aaes-Jorgensen, A., Molin, S., and Tolker-Nielsen, T. (2003b). Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants. Mol Microbiol 48, 1511-1524. Ko, J., Ryu, K.-S., Kim, H.-J., Shin, J.-S., Lee, J.-O., Cheong, C., and Choi, B.-S. (2010). Structure of PP4397 reveals the molecular basis for different c-di-GMP binding modes by PilZ domain proteins. J Mol Biol 398, 97-110. Krasteva, P. V., Fong, J. C. N., Shikuma, N. J., Beyhan, S., Navarro, M. V. A. S., Yildiz, F. H., and Sondermann, H. (2010). Vibrio cholerae VpsT regulates matrix production and motility by directly sensing cyclic di-GMP. Science 327, 866-868. Krissinel, E., and Henrick, K. (2007). Inference of macromolecular assemblies from crystalline state. J Mol Biol 372, 774-797. Kulshina, N., Baird, N. J., and Ferré-D''Amaré, A. R. (2009). Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch. Nat Struct Mol Biol 16, 1212–1217. Leduc, J. L., and Roberts, G. P. (2009). Cyclic di-GMP allosterically inhibits the CRP-like protein (Clp) of Xanthomonas axonopodis pv. citri. J Bacteriol 191, 7121-7122. Li, T.-N., Chin, K.-H., Fung, K.-M., Yang, M.-T., Wang, A. H.-J., and Chou, S.-H. (2011). A novel tetrameric PilZ domain structure from Xanthomonads. PLoS ONE 6, e22036. Li, T.-N., Chin, K.-H., Liu, J.-H., Wang, A. H.-J., and Chou, S.-H. (2009a). XC1028 from Xanthomonas campestris adopts a PilZ domain-like structure without a c-di-GMP switch. Proteins: Structure, Function and Bioinformatics 75, 282-288. Li, T.-N., Chin, K.-H., Shih, H.-L., Wang, A. H.-J., and Chou, S.-H. (2009b). Crystallization and preliminary X-ray diffraction characterization of an essential protein from Xanthomonas campestris that contains a noncanonical PilZ signature motif yet is critical for the Xcc pathogenicity. Acta Crystallogr F65, 1056-1059. Liao, Y.-T., Chin, K.-H., Kuo, W.-T., Chuah, M. L.-C., Liang, Z.-X., and Chou, S.-H. (2012). On the crystallization and preliminary X-ray diffraction characterization of FimXEAL-c-di-GMP and FimXEAL-c-di-GMP-PilZ complexes from Xanthomonas campestris. Acta Crystallogr F68, 301-305. Mattick, J. S. (2002). Type IV pili and twitching motility. Annu Rev Microbiol 56, 289-314. McCarthy, Y., Ryan, R. P., O''donovan, K., He, Y.-Q., Jiang, B.-L., Feng, J.-X., Tang, J.-L., and Dow, J. M. (2008). The role of PilZ domain proteins in the virulence of Xanthomonas campestrris pv. campestris. Mol Plant Path 9, 819-824. McRee, D. E. (1999). XtalView/Xfit - A versatile program for manipulating atomic coordinates and electron density. J Struct Biol 125, 156-165. Minasov, G., Padavattan, S., Shuvalova, L., Brunzelle, J. S., Miller, D. J., Baslé, A., Massa, C., Collart, F. R., Schirmer, T., and Anderson, W. F. (2009). Crystal structures of YkuI and its complex with second messenger cyclic di-GMP suggest catalytic mechanism of phosphodiester bond cleavage by EAL domains. J Biol Chem 284, 13174-13184. Navarro, M. V. A. S., De, N., Bae, N., Wang, Q., and Sondermann, H. (2009). Structural analysis of the GGDEF-EAL domain-containing c-di-GMP receptor FimX. Structure 17, 1104-1116. Navarro, M. V. A. S., Newell, P. D., Krasteva, P. V., Chatterjee, D., Madden, D. R., O''Toole, G. A., and Sondermann, H. (2011). Structural Basis for c-di-GMP-Mediated Inside-Out Signaling Controlling Periplasmic Proteolysis. PLoS Biology 9, e1000588. Otwinowski, Z., and Minor, W. (1997). Processing of the X-ray diffraction data collected in oscillation mode. Methods Enzymol 276, 307-326. Passner, J. M., and Steitz, T. A. (1997). The structure of a CAP-DNA complex having two cAMP molecules bound to each monomer. Proc Natl Acad Sci USA 94, 2843-2847. Paul, K., Nieto, V., Carlquist, W. C., Blair, D. F., and Harshey, R. M. (2010). The c-di-GMP binding protein YcgR controls flagellar motor direction and speed to affect chemotaxis by a "backstop brake" mechanism. Mol Cell 38, 128-139. Qi, Y., Chuah, M. L.-C., Dong, X., Xie, K., Luo, Z., Tang, K., and Liang, Z.-X. (2011). Binding of cyclic diguanylate in the non-catalytic EAL domain of FimX induces a long-range conformational change. J Biol Chem 286, 2910-2917. Rao, F., Pasunooti, S., Ng, Y., Zhuo, W., Lim, L., Liu, A. W., and Liang, Z.-X. (2009). Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase. Analytical Biochem 389, 138-142. Romling, U. (2011). Cyclic di-GMP, an established secondary messenger still speeding up. Environmental Microbiology, doi:10.1111/j.1462-2920.2011.02671.x. Romling, U., and Amikam, D. (2006a). Cyclic di-GMP as a second messenger. Curr Opin Microbiology 9, 218-228. Romling, U., and Amikam, D. (2006b). Cyclic di-GMP as a second messenger. Cur Opin Microbiology 9, 218-228. Romling, U., Gomelsky, M., and Galperin, M. Y. (2005). C-di-GMP: the dawning of a novel bacterial signalling system. Mol Microbiol 57, 629-639. Ryan, R. P., Fouhy, Y., Lucey, J. F., Crossman, L. C., Spiro, S., He, Y.-W., Zhang, L.-H., Heeb, S., Camara, M., Williams, P., and Dow, J. M. (2006). Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proc Natl Acad Sci USA 103, 6712-6717. Ryan, R. P., Tolker-Nielsen, T., and Dow, J. M. (2012). When the PilZ don''t work: effectors for cyclic di-GMP action in bacteria. Trends in Microbiol in press. Savvides, S. (2007). Secretion superfamily ATPases swing big. Structure 15, 255-257. Schirmer, T., and Jenal, U. (2009). Structural and mechanistic determinants of c-di-GMP signalling. Nat Rev Microbiol 7, 724-725. Simm, R., Morr, M., Kader, A., Nimtz, M., and Romling, U. (2004). GGDEF and EAL domains inverslely regulate cyclic di-GMP levels and transition from sessility to motility. Mol Microbiol 53, 1123-1134. Slater, H., Alvarez-Morales, A., Barber, C. E., Daniels, M. J., and Dow, J. M. (2000). A two-component system involving an HD-GYP domain protein links cell-cell signalling to pathogenicity gene expression in Xanthomonas campestris. Mol Microbiol 38, 986-1003. Smith, K. D., Lipchock, S. V., Ames, T. D., Wang, J., Breaker, R. R., and Strobel1, S. A. (2009). Structural basis of ligand binding by a c-di-GMP riboswitch. Nat Struct Mol Biol 16, 1218-1223. Smith, K. D., Shanahan, C. A., Moore, E. L., Simon, A. C., and Strobel, S. A. (2011). Structural basis of differential ligand recognition by two classes of bis-(3''-5'')-cyclic dimeric guanosine monophosphate-binding riboswitches. Proc Natl Acad Sci USA 108, 7757-7762. Sondermann, H., Shikuma, N. J., and Yildiz, F. H. (2011). You''ve come a long way: c-di-GMP signaling. Cur Opin Microbiology 15, doi:10.1016/j.mib.2011.1012.1008. Sterner, R., and Hocker, B. (2005). Catalytic versatility, stability, and evolution of the (ba)8-barrel enzyme fold. Chem Rev 105, 4038-4055. Tal, R., Wong, H. C., Calhoon, R., Gelfand, D., Fear, A. L., Volman, G., Mayer, R., Ross, P., Amikam, D., Weinhouse, H., et al. (1988). Three cdg Operons Control Cellular Turnover of Cyclic Di-GMP in Acetobacter xylinum: Genetic Organization and Occurrence of Conserved Domains in Isoenzymes. J Bacteriol 180, 4416-4425. Tao, F., He, Y.-W., Wu, D.-H., Swarup, S., and 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. Tchigvintsev, A., Xu, X., Singer, A., Chang, C., Brown, G., Proudfoot, M., Cui, H., Flick, R., Anderson, W. F., Joachimiak, A., et al. (2010). Structural insight into the mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases. J Mol Biol 402, 524-538. Thormann, K. M., and Paulick, A. (2010). Tuning the flagellar motor. Microbiology 156, 1275–1283. Tischler, A. D., and Camilli, A. (2004). Cyclic diguanylate (c-di-GMP) regulates Vibrio cholerae biofilm formation. Mol Microbiol 53, 857-869. Tuckerman, J. R., Gonzalez, G., and Gilles-Gonzalez, M.-A. (2011). Cyclic di-GMP activation of polynucleotide phosphorylase signal-dependent RNA processing. J Mol Biol 407, 622-639. Wallace, A. C., Laskowski, R. A., and Thornton, J. M. (1995). LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Engineering 8, 127-134. Wang, J., Zhou, J., Donaldson, G. P., Nakayama, S., Yan, L., Lam, Y. F., Lee, W. T., and Sintim, H. O. l. (2010). Conservative Change to the Phosphate Moiety of Cyclic Diguanylic Monophosphate Remarkably Affects Its Polymorphism and Ability To Bind DGC, PDE, and PilZ Proteins. J Am Chem Soc 133, 9320-9330. Wassmann, P., Chan, C., Paul, R., Beck, A., Heerklotz, H., Jenal, U., and Schirmer, T. (2007). Structure of BeF3- -modified response regulator PleD: Implications for diguanylate cyclase activation, catalysis, and feedback inhibition. Structure 15, 915-927. Winn, M. D., Isupovb, M. N., and Murshudo, G. N. (2000). Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr D57, 122-133. Wu, Y.-Y., Chin, K.-H., Chou, C.-C., Lee, C.-C., Shr, H.-L., Lyu, P.-C., Wang, A. H.-J., and Chou, S.-H. (2005). The Cloning, purification, crystallization, and preliminary X-ray crystallographic analysis of XC847, a 3''-5'' oligoribonuclease from Xanthomonas campestris. Acta Crystallogr F61, 902-905. Yang, C.-Y., Chin, K.-H., Chuah, M. L.-C., Liang, Z.-X., Wang, A. H.-J., and Chou, S.-H. (2011). On the Structure and Inhibition of a GGDEF Diguanylate Cyclase Complexed with (c-di-GMP)2 at Active Site. Acta Crystallogr D67, 997-1008. Yeo, H., Savvides, S., Herr, E., Lanka, E., and Waksman, G. (2000). Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system. Mol Cell 6, 1461-1472. Zhang, Z., Kim, S., Gaffney, B. L., and Jones, R. A. (2006). Polymorphism of the signaling molecule c-di-GMP. J Am Chem Soc 128, 7015-7024.
c-di-GMP是細菌中一個重要的訊息傳遞分子,參與調控細胞中許多重要的生物功能,包括毒性因子的表達、生物膜的合成以及細胞的運動…等,而其濃度則由GGDEF 和EAL domain負責調節;GGDEF domain具有 Diguanylate Cyclases (DGC) 活性以合成c-di-GMP,而EAL domain 則具有 Phosphodiesterases (PDE)活性以分解c-di-GMP。在先前的文獻中,已得知當細菌中c-di-GMP濃度高時,非典型第二型PilZ 蛋白會與FimX和PilB結合形成複合體 (complex) 進而調控細胞第四型分泌機制 (T4P) 。利用生物資訊方法找尋FimX基因在Xanthomonas campestris中被註解的基因號碼為XC2112 ,而XccFimX (Xc2112) 由Rec,Pas-like,GGDEF和EAL 四個domains所組成。在本研究中,我們利用X-ray的晶體繞射技術解析了Xanthomonas campestris 中XccFimXEAL-c-di-GMP XccFimXEAL-c-di-GMP-XccPilZ1028之複合體結構。觀察XccFimXEAL-c-di-GMP複合體之三度空間結構,我們發現XccFimXEAL-c-di-GMP為一二聚體。利用ITC測試XccFimXEAL與c-di-GMP的解離常數 (KD)為4.2 x 10-7,得知XccFimXEAL不具有Phosphodiesterases活性,且與c-di-GMP具有很大的結合力。另外,觀察XccFimXEAL-c-di-GMP-XccPilZ1028複合體之三度空間結構,我們發現XccFimXEAL會與c-di-GMP和XccPilZ1028形成穩定的四聚體。此外,我們同時也利用ITC和膠体過濾法 (Gel filtration) 證實,XccFimXEAL (474-704)和XccPilZ1028在c-di-GMP不存在的情況之下個別為一單體,一旦c-di-GMP出現,XccFimXEAL (474-704)就會與c-di-GMP和XccPilZ1028結合成穩定四聚體。綜合以上的結果,我們初步了解細胞中c-di-GMP調控第四型分泌機制 (T4P)的方式。

c-di-GMP is a key signal molecule involved in regulating many important biological functions in bacteria, such as surface motility, biofilm formation and pathogenicity. GGDEF-domain containing diguanylate cyclases (DGC) and EAL-domain containing phosphodiesterases (PDE) are identified as the enzymes controlling the synthesis and degradation of c-di-GMP, respectively. Recent data indicate that T4P biogenesis is initiated via interaction of a non-canonical type II PilZ protein with the FimX and PilB ATPase under high c-di-GMP concentration. It is thus essential to determine the FimXEAL–PilZ complex structure to see how the binding of c-di-GMP with FimXEAL domain induces conformational change of the bound non-canonical PilZ domain, which may transmit information to PilB or PilT to control T4P function. We now report two novel conformations for monomeric c-di-GMP in the XccFimXEAL-c-di-GMP and XccFimXEAL-c-di-GMP-XccPilZ complex structures and the unique interaction mode of type II PilZ domain with c-di-GMP. We show that XccFimXEAL is a degenerate c-di-GMP phosphodiesterase (PDE), and the wild type XccFimXEAL can bind c-di-GMP with a KD in the μM range as measured by the ITC method. We also demonstrate that c-di-GMP is indispensable for the stable formation of type II XccPilZ1028-XccFimXEAL complex, which is evidenced by a variety of biophysical methods including ITC and gel filtration chromatography. Taken together, the results represent a first step toward understanding of how T4P biogenesis is controlled by c-di-GMP at molecular level.
其他識別: U0005-2307201216011800
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