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標題: c-di-GMP如何增進LtmA與其同源DNA結合的結構研究
How c-di-GMP enhances the binding of LtmA toward its cognate DNA sequences
作者: 胡惠喻
Hui-Yu Hu
關鍵字: 二級訊息傳遞分子
transcription factor
secondary messenger
引用: 1. Ross P, et al. (1987) Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325(6101):279-281. 2. Chan C, et al. (2004) Structural basis of activity and allosteric control of diguanylate cyclase. Proceedings of the National Academy of Sciences of the United States of America 101(49):17084-17089. 3. Wassmann P, et al. (2007) Structure of BeF3- -modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition. Structure 15(8):915-927. 4. De N, et al. (2008) Phosphorylation-independent regulation of the diguanylate cyclase WspR. PLoS biology 6(3):e67. 5. 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. The Journal of biological chemistry 280(35):30829-30837. 6. Schmidt AJ, Ryjenkov DA, & Gomelsky M (2005) The ubiquitous protein domain EAL is a cyclic diguanylate-specific phosphodiesterase: enzymatically active and inactive EAL domains. Journal of bacteriology 187(14):4774-4781. 7. Ryan RP, et al. (2006) Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover. Proceedings of the National Academy of Sciences of the United States of America 103(17):6712-6717. 8. Sondermann H, Shikuma NJ, & Yildiz FH (2012) You've come a long way: c-di-GMP signaling. Current opinion in microbiology 15(2):140-146. 9. Cotter PA & Stibitz S (2007) c-di-GMP-mediated regulation of virulence and biofilm formation. Current opinion in microbiology 10(1):17-23. 10. Ryan RP, et al. (2007) Cyclic di-GMP signalling in the virulence and environmental adaptation of Xanthomonas campestris. Molecular microbiology 63(2):429-442. 11. Tamayo R, Pratt JT, & Camilli A (2007) Roles of cyclic diguanylate in the regulation of bacterial pathogenesis. Annual review of microbiology 61:131-148. 12. Tamayo R, Schild S, Pratt JT, & Camilli A (2008) Role of cyclic Di-GMP during el tor biotype Vibrio cholerae infection: characterization of the in vivo-induced cyclic Di-GMP phosphodiesterase CdpA. Infection and immunity 76(4):1617-1627. 13. Lai TH, Kumagai Y, Hyodo M, Hayakawa Y, & Rikihisa Y (2009) The Anaplasma phagocytophilum PleC histidine kinase and PleD diguanylate cyclase two-component system and role of cyclic Di-GMP in host cell infection. Journal of bacteriology 191(3):693-700. 14. Lee VT, et al. (2007) A cyclic-di-GMP receptor required for bacterial exopolysaccharide production. Molecular microbiology 65(6):1474-1484. 15. Monds RD, Newell PD, Gross RH, & O'Toole GA (2007) Phosphate-dependent modulation of c-di-GMP levels regulates Pseudomonas fluorescens Pf0-1 biofilm formation by controlling secretion of the adhesin LapA. Molecular microbiology 63(3):656-679. 16. Nakhamchik A, Wilde C, & Rowe-Magnus DA (2008) Cyclic-di-GMP regulates extracellular polysaccharide production, biofilm formation, and rugose colony development by Vibrio vulnificus. Applied and environmental microbiology 74(13):4199-4209. 17. Girgis HS, Liu Y, Ryu WS, & Tavazoie S (2007) A comprehensive genetic characterization of bacterial motility. PLoS genetics 3(9):1644-1660. 18. Wolfe AJ & Visick KL (2008) Get the message out: cyclic-Di-GMP regulates multiple levels of flagellum-based motility. Journal of bacteriology 190(2):463-475. 19. Amikam D & Galperin MY (2006) PilZ domain is part of the bacterial c-di-GMP binding protein. Bioinformatics 22(1):3-6. 20. Merighi M, Lee VT, Hyodo M, Hayakawa Y, & Lory S (2007) The second messenger bis-(3'-5')-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa. Molecular microbiology 65(4):876-895. 21. Pratt JT, Tamayo R, Tischler AD, & Camilli A (2007) PilZ domain proteins bind cyclic diguanylate and regulate diverse processes in Vibrio cholerae. The Journal of biological chemistry 282(17):12860-12870. 22. Wilksch JJ, et al. (2011) MrkH, a novel c-di-GMP-dependent transcriptional activator, controls Klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression. PLoS pathogens 7(8):e1002204. 23. Sudarsan N, et al. (2008) Riboswitches in eubacteria sense the second messenger cyclic di-GMP. Science 321(5887):411-413. 24. Hickman JW & Harwood CS (2008) Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Molecular microbiology 69(2):376-389. 25. Krasteva PV, et al. (2010) Vibrio cholerae VpsT regulates matrix production and motility by directly sensing cyclic di-GMP. Science 327(5967):866-868. 26. Chin KH, et al. (2010) The cAMP receptor-like protein CLP is a novel c-di-GMP receptor linking cell-cell signaling to virulence gene expression in Xanthomonas campestris. Journal of molecular biology 396(3):646-662. 27. Fazli M, et al. (2011) The CRP/FNR family protein Bcam1349 is a c-di-GMP effector that regulates biofilm formation in the respiratory pathogen Burkholderia cenocepacia. Molecular microbiology 82(2):327-341. 28. He YW, et al. (2007) Xanthomonas campestris cell-cell communication involves a putative nucleotide receptor protein Clp and a hierarchical signalling network. Molecular microbiology 64(2):281-292. 29. Leduc JL & Roberts GP (2009) Cyclic di-GMP allosterically inhibits the CRP-like protein (Clp) of Xanthomonas axonopodis pv. citri. Journal of bacteriology 191(22):7121-7122. 30. Tao F, He YW, Wu DH, Swarup S, & Zhang LH (2010) The cyclic nucleotide monophosphate domain of Xanthomonas campestris global regulator Clp defines a new class of cyclic di-GMP effectors. Journal of bacteriology 192(4):1020-1029. 31. Li W & He ZG (2012) LtmA, a novel cyclic di-GMP-responsive activator, broadly regulates the expression of lipid transport and metabolism genes in Mycobacterium smegmatis. Nucleic acids research 40(22):11292-11307. 32. Tuckerman JR, Gonzalez G, & Gilles-Gonzalez MA (2011) Cyclic di-GMP activation of polynucleotide phosphorylase signal-dependent RNA processing. Journal of molecular biology 407(5):633-639. 33. Navarro MV, De N, Bae N, Wang Q, & Sondermann H (2009) Structural analysis of the GGDEF-EAL domain-containing c-di-GMP receptor FimX. Structure 17(8):1104-1116. 34. Navarro MV, et al. (2011) Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis. PLoS biology 9(2):e1000588. 35. Benach J, et al. (2007) The structural basis of cyclic diguanylate signal transduction by PilZ domains. The EMBO journal 26(24):5153-5166. 36. Chou SH & Galperin MY (2015) Diversity of c-di-GMP-binding proteins and mechanisms. Journal of bacteriology. 37. Reyrat JM & Kahn D (2001) Mycobacterium smegmatis: an absurd model for tuberculosis? Trends in microbiology 9(10):472-474. 38. Snapper SB, Melton RE, Mustafa S, Kieser T, & Jacobs WR, Jr. (1990) Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Molecular microbiology 4(11):1911-1919. 39. Gupta K, Kumar P, & Chatterji D (2010) Identification, activity and disulfide connectivity of C-di-GMP regulating proteins in Mycobacterium tuberculosis. PloS one 5(11):e15072. 40. Kumar M & Chatterji D (2008) Cyclic di-GMP: a second messenger required for long-term survival, but not for biofilm formation, in Mycobacterium smegmatis. Microbiology 154(Pt 10):2942-2955. 41. Tovar K, Ernst A, & Hillen W (1988) Identification and nucleotide sequence of the class E tet regulatory elements and operator and inducer binding of the encoded purified Tet repressor. Molecular & general genetics : MGG 215(1):76-80. 42. Matsuoka H, Hirooka K, & Fujita Y (2007) Organization and function of the YsiA regulon of Bacillus subtilis involved in fatty acid degradation. The Journal of biological chemistry 282(8):5180-5194. 43. Grkovic S, Hardie KM, Brown MH, & Skurray RA (2003) Interactions of the QacR multidrug-binding protein with structurally diverse ligands: implications for the evolution of the binding pocket. Biochemistry 42(51):15226-15236. 44. Schumacher MA, et al. (2001) Structural mechanisms of QacR induction and multidrug recognition. Science 294(5549):2158-2163. 45. Yang S, et al. (2013) Structural basis for interaction between Mycobacterium smegmatis Ms6564, a TetR family master regulator, and its target DNA. The Journal of biological chemistry 288(33):23687-23695. 46. Deng W, Li C, & Xie J (2013) The underling mechanism of bacterial TetR/AcrR family transcriptional repressors. Cellular signalling 25(7):1608-1613. 47. Ramos JL, et al. (2005) The TetR family of transcriptional repressors. Microbiology and molecular biology reviews : MMBR 69(2):326-356. 48. Itou H, et al. (2005) The CGL2612 protein from Corynebacterium glutamicum is a drug resistance-related transcriptional repressor: structural and functional analysis of a newly identified transcription factor from genomic DNA analysis. The Journal of biological chemistry 280(46):38711-38719. 49. Le TB, et al. (2011) Structures of the TetR-like simocyclinone efflux pump repressor, SimR, and the mechanism of ligand-mediated derepression. Journal of molecular biology 408(1):40-56. 50. Miller DJ, Zhang YM, Subramanian C, Rock CO, & White SW (2010) Structural basis for the transcriptional regulation of membrane lipid homeostasis. Nature structural & molecular biology 17(8):971-975. 51. Orth P, Schnappinger D, Hillen W, Saenger W, & Hinrichs W (2000) Structural basis of gene regulation by the tetracycline inducible Tet repressor-operator system. Nature structural biology 7(3):215-219. 52. Sawai H, Yamanaka M, Sugimoto H, Shiro Y, & Aono S (2012) Structural basis for the transcriptional regulation of heme homeostasis in Lactococcus lactis. The Journal of biological chemistry 287(36):30755-30768. 53. Schumacher MA, et al. (2002) Structural basis for cooperative DNA binding by two dimers of the multidrug-binding protein QacR. The EMBO journal 21(5):1210-1218. 54. Cuthbertson L & Nodwell JR (2013) The TetR family of regulators. Microbiology and molecular biology reviews : MMBR 77(3):440-475. 55. Ettner N, et al. (1995) Proximity mapping of the Tet repressor-tetracycline-Fe2+ complex by hydrogen peroxide mediated protein cleavage. Biochemistry 34(1):22-31. 56. Alguel Y, et al. (2007) Crystal structures of multidrug binding protein TtgR in complex with antibiotics and plant antimicrobials. Journal of molecular biology 369(3):829-840. 57. Hernandez A, et al. (2009) Structural and functional analysis of SmeT, the repressor of the Stenotrophomonas maltophilia multidrug efflux pump SmeDEF. The Journal of biological chemistry 284(21):14428-14438. 58. Willems AR, et al. (2008) Crystal structures of the Streptomyces coelicolor TetR-like protein ActR alone and in complex with actinorhodin or the actinorhodin biosynthetic precursor (S)-DNPA. Journal of molecular biology 376(5):1377-1387. 59. Bellinzoni M, et al. (2009) Structural plasticity and distinct drug-binding modes of LfrR, a mycobacterial efflux pump regulator. Journal of bacteriology 191(24):7531-7537. 60. Le TB, et al. (2009) Coupling of the biosynthesis and export of the DNA gyrase inhibitor simocyclinone in Streptomyces antibioticus. Molecular microbiology 72(6):1462-1474. 61. Lei HT, et al. (2011) Crystal structures of CmeR-bile acid complexes from Campylobacter jejuni. Protein science : a publication of the Protein Society 20(4):712-723. 62. Agari Y, Agari K, Sakamoto K, Kuramitsu S, & Shinkai A (2011) TetR-family transcriptional repressor Thermus thermophilus FadR controls fatty acid degradation. Microbiology 157(Pt 6):1589-1601. 63. Frenois F, Engohang-Ndong J, Locht C, Baulard AR, & Villeret V (2004) Structure of EthR in a ligand bound conformation reveals therapeutic perspectives against tuberculosis. Molecular cell 16(2):301-307. 64. Beckers G, et al. (2005) Regulation of AmtR-controlled gene expression in Corynebacterium glutamicum: mechanism and characterization of the AmtR regulon. Molecular microbiology 58(2):580-595. 65. Bernhardt TG & de Boer PA (2005) SlmA, a nucleoid-associated, FtsZ binding protein required for blocking septal ring assembly over Chromosomes in E. coli. Molecular cell 18(5):555-564. 66. Christen S, et al. (2006) Regulation of the Dha operon of Lactococcus lactis: a deviation from the rule followed by the Tetr family of transcription regulators. The Journal of biological chemistry 281(32):23129-23137. 67. Cho H & Bernhardt TG (2013) Identification of the SlmA active site responsible for blocking bacterial cytokinetic ring assembly over the chromosome. PLoS genetics 9(2):e1003304. 68. Aslanidis C & de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic acids research 18(20):6069-6074. 69. Pryor EE, Jr., Wozniak DJ, & Hollis T (2012) Crystallization of Pseudomonas aeruginosa AmrZ protein: development of a comprehensive method for obtaining and optimization of protein-DNA crystals. Acta crystallographica. Section F, Structural biology and crystallization communications 68(Pt 8):985-993. 70. Otwinowski Z, and Minor, W. (1997) Processing of the X-ray diffraction data collected in scillation mode. Methods Enzymol 276:307-326. 71. Pahler A, Smith JL, & Hendrickson WA (1990) A probability representation for phase information from multiwavelength anomalous dispersion. Acta crystallographica. Section A, Foundations of crystallography 46 ( Pt 7):537-540. 72. Terwilliger TC (2000) Maximum-likelihood density modification. Acta crystallographica. Section D, Biological crystallography 56(Pt 8):965-972. 73. McRee DE (1999) XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. Journal of structural biology 125(2-3):156-165. 74. Adams PD, et al. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta crystallographica. Section D, Biological crystallography 66(Pt 2):213-221. 75. Revzin A (1989) Gel electrophoresis assays for DNA-protein interactions. BioTechniques 7(4):346-355. 76. Rodgers JT, Patel P, Hennes JL, Bolognia SL, & Mascotti DP (2000) Use of biotin-labeled nucleic acids for protein purification and agarose-based chemiluminescent electromobility shift assays. Analytical biochemistry 277(2):254-259. 77. Chau YP & Lu KS (1995) Investigation of the blood-ganglion barrier properties in rat sympathetic ganglia by using lanthanum ion and horseradish peroxidase as tracers. Acta anatomica 153(2):135-144. 78. Frehland E, Kreikenbohm R, & Pohl WG (1982) Steady-state fluorescence polarization in planar lipid membranes. Experimental and theoretical analysis of the fluorophores 8-anilino-1-naphthalenesulfonate, 1,6-Diphenyl-1,3,5-hexatriene, dansyllysine-valinomycin and n-(9-anthroyloxy) fatty acids. Biophysical chemistry 15(1):73-86. 79. Holm L & Sander C (1995) Dali: a network tool for protein structure comparison. Trends in biochemical sciences 20(11):478-480. 80. Fujihashi M, et al. (2014) Structural characterization of a ligand-bound form of Bacillus subtilis FadR involved in the regulation of fatty acid degradation. Proteins 82(7):1301-1310. 81. Carette X, et al. (2012) Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands. Nucleic acids research 40(7):3018-3030. 82. Gu R, et al. (2007) Crystal structure of the transcriptional regulator CmeR from Campylobacter jejuni. Journal of molecular biology 372(3):583-593. 83. Li M, et al. (2007) Crystal structure of the transcriptional regulator AcrR from Escherichia coli. Journal of molecular biology 374(3):591-603.
摘要: 二級訊息傳遞分子cyclic-di-GMP (CDG)廣泛存在於細菌中,並參與調控許多重要的生物功能,包括致病性因子的產生、生物膜(biofilm)的合成以及細菌的移動。透過調控轉錄因子的活性,細菌能夠一次活化或抑制一群基因的表現。前人研究發現,許多調控細菌中重要生理活性的轉錄因子為CDG的受體蛋白,而CDG的結合會活化或抑制這些轉錄因子的活性。然而,目前對CDG是如何調控這些轉錄因子的詳細分子機制仍然不是很清楚。在2012年的文獻中指出恥垢分枝桿菌(Mycobacterium smegmatis)中的轉錄因子LtmA為CDG受體,此蛋白能辨識特定幾種脂質運輸和代謝基因的啟動子,當CDG結合到LtmA時,可增強此蛋白結合到其目標基因的能力。 為了瞭解CDG是如何影響LtmA的構形並增強其結合DNA的能力,本研究針對LtmA蛋白進行不同長度片段的構築及大量表現,並利用ITC及EMSA assay來研究,同時我們利用X-ray晶體繞射實驗來解析蛋白質結構,在養晶過程中加入CDG和/或DNA進行共結晶條件的篩選。最初有獲得LtmA9-184晶體且收到解析度為3.0 Å的X-ray繞射數據。目前已獲得native和SeMet-labeled LtmA9-184-CDG的晶體,且分別收到解析度為2.3和2.1 Å的X-ray繞射數據。接著利用異常散射(anomalous diffraction)解決相位角的問題,並成功解析出結構。從結構中得知,LtmA包含N端的DNA結合域(DNA-binding domain)和C端的配體結合域(ligand-binding domain),屬於類TetR家族的一員。然而,電子雲密度圖中卻無法偵測到CDG的存在。此外, 在LtmA與DNA的複合體結晶中,也收到解析度為2.6-Å的繞射數據,並順利解出結構,但也無法偵測到DNA的電子密度。未來將利用浸泡(soaking)或種晶(seeding)的方式嘗試解決配體問題,並且繼續篩選不同長度的DNA以期能獲得更高解析度的繞射數據。
Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP, CDG) is an important bacterial secondary messenger that is involved in the regulation of many critical processes including motility, biofilm formation and virulence. CDG exerts its regulatory function by directly binding to several types of receptor. A genetic screen in the non-pathogenic microorganism Mycobacterium smegmatis identified LtmA as a new CDG-responsive regulator, which is involved in lipid transport and metabolism. CDG could directly bind to LtmA and enhance its binding ability to its target gene. However, the reason for this enhancement is unclear. In addition, LtmA seems to lack the previously reported CDG binding motifs. To elucidate how CDG binds to LtmA and enhance its DNA-binding ability, several structural and functional assays have been conducted. ITC and EMSA assay were carried out to detect binding between CDG-LtmA and DNA-LtmA. Initially we obtained the apo-form LtmA9-184 crystal and collected its X-ray diffraction data, which reached to a resolution of 3.0 Å. Currently, we have also obtained the native and SeMet-labeled co-crystals of LtmA with CDG. The X-ray diffraction data of native and SeMet-labeled co-crystals were collected to a resolution of 2.3 and 2.1 Å , respectively, and the initial structure of LtmA has been solved by SAD method (single wavelength anomalous dispersion). The results showed that LtmA contains a N-terminal DNA-binding domain and a C-terminal ligand-binding domain, which are conserved in the TetR superfamily. However, we were unable to detect the presence of CDG in the electron density map. In addition, although we could obtain the crystals of LtmA in complex with DNA/CDG and collected their diffraction data to a 2.6 Å, we were unable to detect the presence of DNA/CDG in the electron density map. In the future, we will use soaking method at a higher ligand/protein ratio to obtain the LtmA-CDG co-crystal, and continue to screening DNA targets of varying-length for obtaining the LtmA-DNA-CDG ternary complex co-crystal of higher quality.
文章公開時間: 2018-08-21
Appears in Collections:生物化學研究所



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