EnvZ / OmpR兩成分系統控制了兩種膜孔蛋白OmpF和OmpC的表達，它們調節大腸桿菌內的摩爾滲透壓濃度。為了引起這種適應反應，EnvZ在信號轉導過程中將OmpR在N末端受體域（OmpRn）中保守的天門冬醯胺酸磷酸化。磷酸化的OmpRn會誘導OmpR C端去氧核糖核酸結合域（OmpRc）的構型改變，進而刺激去氧核糖核酸啟動子的識別，結合和活化轉錄。此外，OmpR還可以調節大腸桿菌或其他病原體中許多的持家基因和致病力基因，因此對於細菌的發病機制影響重大。然而對於 OmpR的構型或OmpRc-去氧核糖核酸的結合機制目前仍不清楚。在本研究中，我們解析了OmpRc與OmpF去氧核糖核酸啟動子F1區域的複合體晶體結構。並發現OmpRc獨特的結構域交換結構，在不同的結晶條件下均可觀察到。結構分析顯示，只有以傳統單體形式頭尾方向結合的OmpRc同源二聚體能結合其同源去氧核糖核酸，而結構域交換構型的OmpRc二聚體形式不能結合其同源去氧核糖核酸。我們還發現在結晶結構中，OmpR與特定序列的去氧核糖核酸鹼基有特異性接觸，這顯示OmpR可以作用在多個啟動子上。透過核磁共振光譜儀，螢光偏振和熱穩定性實驗等實驗結果，我們發現未磷酸化的全長OmpR也可以與其去氧核糖核酸啟動子結合，但與磷酸化的全長OmpR或單獨的OmpRc相比，結合親和力較弱。此發現證實磷酸化可以僅增強去氧核糖核酸的結合但不是必需的。此外，我們觀察到 OmpRc會以不對稱方式形成弱二聚體，這顯示未磷酸化OmpRc的去氧核糖核酸結合結構域之間可能具有弱相互作用，因此全長的OmpR可能需要磷酸化或與去氧核糖核酸結合才能正確的形成二聚體。整體而言，本研究展示了全長OmpR在水溶液中的性質, OmpRc獨特的結構域交換二聚狀態和OmpRc-去氧核糖核酸複合物中的二聚體界面。這些知識不僅讓我們對於OmpR的調節作用有更一步的理解，也能用於找尋可能的製藥標地。

EnvZ/OmpR two-component system controls the expression of two membrane porin proteins OmpF and OmpC that regulate the osmolarity levels in Escherichia coli. To elicit this adaptive response, EnvZ phosphorylates OmpR (OmpR-FL) at its conserved Asp residue in the N-terminal receiver domain (OmpRn) during signal transduction. Phosphorylation of OmpRn induces conformational changes in OmpR C-terminal DNA-binding domain (OmpRc) that further stimulates promoter DNA recognition, binding and transcription activation. Apart from that, OmpR is known to regulate many other housekeeping and virulence genes in E. coli and other pathogens and thus is highly responsible for bacterial pathogenesis. In spite of such biological importance, structural information of the OmpRc−DNA binding mechanism is still elusive. In our study, the crystal structure of OmpRc bound to the F1 region of ompF promoter DNA was determined. We also found that OmpRc in the apo-form has a unique domain-swapped structure that was observed in different crystallization conditions. Structural investigation showed that OmpRc could bind to the DNA only in the head-to-tail orientation of a homodimer formed by its traditional monomeric form but not by domain-swapped dimeric form. Furthermore, in the crystal structure, it was seen that DNA sequence-specific contacts of OmpRc with DNA bases were few upon DNA binding, suggesting that OmpR can be functional on multiple promoters. Using several biophysical examinations, such as NMR, thermal stability experiments, and fluorescent polarization we observed that unphosphorylated OmpR-FL could also bind to its promoter DNA, but with a weaker binding affinity compared to the phosphorylated OmpR-FL and OmpRc alone. This finding approves that phosphorylation may not be required but enhance DNA binding. Besides, we observed that OmpRc forms a weak dimer asymmetrically, suggesting that the DNA-binding domain of unphosphorylated OmpR may have weak interactions and therefore OmpR-FL may require phosphorylation or DNA binding for proper dimer formation.
Taken together, the solution behaviour of OmpR-FL, the unique domain-swapped dimeric state of OmpRc and the dimer interfaces in the OmpRc−DNA complex found in this study not only provide a better understanding of the regulatory role of OmpR but also identify the potential for this "druggable" target.

中文摘要……………………………………………………………...……….....i
Abstract………………………………………………………………...…......iii
Acknowledgement…………………………………………………………......….v
Abbreviations……………………………………………………..………….…vii

Chapter 1. Introduction
1.1 Antimicrobial resistance pathogens…………………………………....2
1.2 Bacterial two-component system………………………………..….…...4
1.1.1 Histidine kinase………………………………….………….........…6
1.1.2 Response regulator……………………………………………......……8
1.3 EnvZ/OmpR two-component system………………………………........…9
1.3.1 EnvZ architecture……………………………………………….....….10
1.3.2 OmpR architecture………………………..……………..……...…...10
1.4 Purpose and outline of this study…………………………….…...…13

Chapter 2. Materials and Methods
2.1 Expression and purification of OmpR-FL, OmpRn and OmpRc……....15
2.2 Preparation of double-stranded DNA…………………………..…..….16
2.3 Protein crystallization and data collection…………………….….16
2.4 Structure determination and refinement……………………………...17
2.5 Circular dichroism (CD) spectroscopy…………………………….…..18
2.6 Fluorescence polarization measurements………………………….....18
2.7. Bio-layer interferometry…………………………………….....…….19
2.8 NMR spectroscopy and data processing…………………………....….20

Chapter 3. Results
3.1 Preparation of recombinant OmpR and its variants…………....….23
3.2 Solution behaviour of OmpR-FL……………………………………..…..23
3.3 Solution Behavior of OmpRc………………………………..……..…...25
3.4 Solution Behavior of OmpRn………………………………..…….......26
3.5 Crystallization trials with OmpR-FL…………………………..….….28
3.6 Crystallization trials with OmpRc-apo form……………………..….29
3.6.1 Crystal structure of OmpRc monomeric apo-form…………...…...29
3.6.2 Crystal structure of OmpRc domain-swapped dimer form……...…30
3.7 Crystal structure of OmpRc−F1-DNA………………………….……...…32
3.7.1 Interactions of OmpRc with DNA…………………………..……...…33
3.7.2 DBD-DBD interface in OmpRc–DNA complex……………..…....…..36
3.8 Biophysical examination of OmpR with DNA……………………...…..37
3.8.1 Promoter recognition by OmpR………………………..…......…….37
3.8.2 Inactive OmpR-FL can bind to promoter DNA…………..……..…..39
3.8.3 Effect of phosphorylation on OmpR−DNA binding…..............40
3.8.4 Investigating intermolecular interactions of OmpRc in solution...42

Chapter 4. Discussion.
4.1 Structural characteristics of OmpR-FL and OmpRc………………....45
4.2 Domain swapping dimeric OmpRc………………………………………....49
4.3 OmpR’s capability to regulate multiple genes………………………50

Figures………………………………………………………………….…53
References…………………………………………………………………94
Appendix……………………………………………………………………99

1. Ann M. Stock, a. Victoria L. Robinson, and P.N. Goudreau, Two-Component Signal Transduction. Annual Review of Biochemistry, 2000. 69(1): p. 183-215.
2. Mascher, T., J.D. Helmann, and G. Unden, Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases. Microbiology and Molecular Biology Reviews, 2006. 70(4): p. 910-938.
3. Capra, E.J. and M.T. Laub, Evolution of Two-Component Signal Transduction Systems. Annual Review of Microbiology, 2012. 66(1): p. 325-347.
4. Wolanin, P.M., P.A. Thomason, and J.B. Stock, Histidine protein kinases: key signal transducers outside the animal kingdom. Genome Biology, 2002. 3(10): p. reviews3013.1.
5. Schaller, G.E., S.-H. Shiu, and Judith P. Armitage, Two-Component Systems and Their Co-Option for Eukaryotic Signal Transduction. Current Biology, 2011. 21(9): p. R320-R330.
6. Wuichet, K., B.J. Cantwell, and I.B. Zhulin, Evolution and phyletic distribution of two-component signal transduction systems. Current Opinion in Microbiology, 2010. 13(2): p. 219-225.
7. Herrou, J., S. Crosson, and A. Fiebig, Structure and function of HWE/HisKA2-family sensor histidine kinases. Current Opinion in Microbiology, 2017. 36: p. 47-54.
8. Sanders, D.A., et al., Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY. J Biol Chem, 1989. 264(36): p. 21770-8.
9. Sanders, D.A., et al., Phosphorylation site of NtrC, a protein phosphatase whose covalent intermediate activates transcription. Journal of Bacteriology, 1992. 174(15): p. 5117-5122.
10. West, A.H. and A.M. Stock, Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem Sci, 2001. 26(6): p. 369-76.
11. Mascher, T., J.D. Helmann, and G. Unden, Stimulus perception in bacterial signal-transducing histidine kinases. Microbiol Mol Biol Rev, 2006. 70(4): p. 910-38.
12. Stock, J.B., A.J. Ninfa, and A.M. Stock, Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev, 1989. 53(4): p. 450-90.
13. Tomomori, C., et al., Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ. Nat Struct Biol, 1999. 6(8): p. 729-34.
14. Bilwes, A.M., et al., Structure of CheA, a signal-transducing histidine kinase. Cell, 1999. 96(1): p. 131-41.
15. Vierstra, R.D. and S.J. Davis, Bacteriophytochromes: new tools for understanding phytochrome signal transduction. Semin Cell Dev Biol, 2000. 11(6): p. 511-21.
16. Alex, L.A. and M.I. Simon, Protein histidine kinases and signal transduction in prokaryotes and eukaryotes. Trends Genet, 1994. 10(4): p. 133-8.
17. Parkinson, J.S. and E.C. Kofoid, Communication modules in bacterial signaling proteins. Annu Rev Genet, 1992. 26: p. 71-112.
18. Sanders, D.A., et al., Phosphorylation site of NtrC, a protein phosphatase whose covalent intermediate activates transcription. J Bacteriol, 1992. 174(15): p. 5117-22.
19. Dubey, B.N., et al., Cyclic di-GMP mediates a histidine kinase/phosphatase switch by noncovalent domain cross-linking. Sci Adv, 2016. 2(9): p. e1600823.
20. Liu, Y., et al., A pH-gated conformational switch regulates the phosphatase activity of bifunctional HisKA-family histidine kinases. Nat Commun, 2017. 8(1): p. 2104.
21. Galperin, M.Y., A census of membrane-bound and intracellular signal transduction proteins in bacteria: bacterial IQ, extroverts and introverts. BMC Microbiol, 2005. 5: p. 35.
22. Galperin, M.Y., Structural classification of bacterial response regulators: diversity of output domains and domain combinations. J Bacteriol, 2006. 188(12): p. 4169-82.
23. Barbieri, C.M., T. Wu, and A.M. Stock, Comprehensive analysis of OmpR phosphorylation, dimerization, and DNA binding supports a canonical model for activation. J Mol Biol, 2013. 425(10): p. 1612-26.
24. Kenney, L.J., Structure/function relationships in OmpR and other winged-helix transcription factors. Curr Opin Microbiol, 2002. 5(2): p. 135-41.
25. Baikalov, I., et al., Structure of the Escherichia coli response regulator NarL. Biochemistry, 1996. 35(34): p. 11053-61.
26. Wurtzel, E.T., M.Y. Chou, and M. Inouye, Osmoregulation of gene expression. I. DNA sequence of the ompR gene of the ompB operon of Escherichia coli and characterization of its gene product. J Biol Chem, 1982. 257(22): p. 13685-91.
27. Cai, S.J. and M. Inouye, EnvZ-OmpR interaction and osmoregulation in Escherichia coli. J Biol Chem, 2002. 277(27): p. 24155-61.
28. Slauch, J.M. and T.J. Silhavy, Genetic analysis of the switch that controls porin gene expression in Escherichia coli K-12. J Mol Biol, 1989. 210(2): p. 281-92.
29. Slauch, J.M., et al., EnvZ functions through OmpR to control porin gene expression in Escherichia coli K-12. J Bacteriol, 1988. 170(1): p. 439-41.
30. Alphen, W.V. and B. Lugtenberg, Influence of osmolarity of the growth medium on the outer membrane protein pattern of Escherichia coli. J Bacteriol, 1977. 131(2): p. 623-30.
31. Dedieu, L., J.M. Pagès, and J.M. Bolla, The omp50 gene is transcriptionally controlled by a temperature-dependent mechanism conserved among thermophilic Campylobacter species. Res Microbiol, 2008. 159(4): p. 270-8.
32. Todt, J.C. and E.J. McGroarty, Acid pH decreases OmpF and OmpC channel size in vivo. Biochem Biophys Res Commun, 1992. 189(3): p. 1498-502.
33. Thomas, A.D. and I.R. Booth, The regulation of expression of the porin gene ompC by acid pH. J Gen Microbiol, 1992. 138(9): p. 1829-35.
34. Nikaido, H. and M. Vaara, Molecular basis of bacterial outer membrane permeability. Microbiol Rev, 1985. 49(1): p. 1-32.
35. Forst, S., et al., Localization and membrane topology of EnvZ, a protein involved in osmoregulation of OmpF and OmpC in Escherichia coli. J Biol Chem, 1987. 262(34): p. 16433-8.
36. Foo, Y.H., et al., Cytoplasmic sensing by the inner membrane histidine kinase EnvZ. Prog Biophys Mol Biol, 2015. 118(3): p. 119-29.
37. Wang, L.C., et al., The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm. Embo j, 2012. 31(11): p. 2648-59.
38. Tanaka, T., et al., NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ. Nature, 1998. 396(6706): p. 88-92.
39. Appleman, J.A., L.L. Chen, and V. Stewart, Probing conservation of HAMP linker structure and signal transduction mechanism through analysis of hybrid sensor kinases. J Bacteriol, 2003. 185(16): p. 4872-82.
40. Toro-Roman, A., T.R. Mack, and A.M. Stock, Structural analysis and solution studies of the activated regulatory domain of the response regulator ArcA: A symmetric dimer mediated by the alpha 4-beta 5-alpha 5 face. Journal of Molecular Biology, 2005. 349(1): p. 11-26.
41. Gao, R., T.R. Mack, and A.M. Stock, Bacterial response regulators: versatile regulatory strategies from common domains. Trends in Biochemical Sciences, 2007. 32(5): p. 225-234.
42. Jeon, Y., et al., Multimerization of phosphorylated and non-phosphorylated ArcA is necessary for the response regulator function of the Arc two-component signal transduction system. J Biol Chem, 2001. 276(44): p. 40873-9.
43. Kondo, H., et al., Escherichia coli positive regulator OmpR has a large loop structure at the putative RNA polymerase interaction site. Nature Structural Biology, 1997. 4(1): p. 28-31.
44. MartinezHackert, E. and A.M. Stock, The DNA-binding domain of OmpR: Crystal structure of a winged helix transcription factor. Structure, 1997. 5(1): p. 109-124.
45. Bourret, R.B., Receiver domain structure and function in response regulator proteins. Current Opinion in Microbiology, 2010. 13(2): p. 142-149.
46. Mattison, K., R. Oropeza, and L.J. Kenney, The linker region plays an important role in the interdomain communication of the response regulator OmpR. Journal of Biological Chemistry, 2002. 277(36): p. 32714-32721.
47. Walthers, D., V. Tran, and L.J. Kenney, Interdomain linkers of homologous response regulators determine their mechanism of action. Journal of Bacteriology, 2003. 185(1): p. 317-324.
48. Robinson, V.L., T. Wu, and A.M. Stock, Structural analysis of the domain interface in DrrB, a response regulator of the OmpR/PhoB subfamily. Journal of Bacteriology, 2003. 185(14): p. 4186-4194.
49. Friedland, N., et al., Domain orientation in the inactive response regulator Mycobacterium tuberculosis MtrA provides a barrier to activation. Biochemistry, 2007. 46(23): p. 6733-6743.
50. Nowak, E., et al., The structural basis of signal transduction for the response regulator PrrA from Mycobacterium tuberculosis. Journal of Biological Chemistry, 2006. 281(14): p. 9659-9666.
51. King-Scott, J., et al., The structure of a full-length response regulator from Mycobacterium tuberculosis in a stabilized three-dimensional domain-swapped, activated state. J Biol Chem, 2007. 282(52): p. 37717-29.
52. Buckler, D.R., Y.C. Zhou, and A.M. Stock, Evidence of intradomain and interdomain flexibility in an OmpR/PhoB homolog from Thermotoga maritima. Structure, 2002. 10(2): p. 153-164.
53. Lou, Y.C., et al., Structure and dynamics of polymyxin-resistance-associated response regulator PmrA in complex with promoter DNA. Nat Commun, 2015. 6: p. 8838.
54. Narayanan, A., et al., An asymmetric heterodomain interface stabilizes a response regulator-DNA complex. Nature Communications, 2014. 5.
55. Rhee, J.E., et al., Amino acids important for DNA recognition by the response regulator OmpR. Journal of Biological Chemistry, 2008. 283(13): p. 8664-8677.
56. Maris, A.E., et al., The response regulator OmpR oligomerizes via beta-sheets to form head-to-head dimers. J Mol Biol, 2005. 350(5): p. 843-56.
57. Otwinowski, Z. and W. Minor, Processing of X-ray diffraction data collected in oscillation mode. Macromolecular Crystallography, Pt A, 1997. 276: p. 307-326.
58. McCoy, A.J., et al., Phaser crystallographic software. Journal of Applied Crystallography, 2007. 40: p. 658-674.
59. Li, Y.C., et al., Structural dynamics of the two-component response regulator RstA in recognition of promoter DNA element. Nucleic Acids Res, 2014. 42(13): p. 8777-88.
60. Adams, P.D., et al., PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D-Biological Crystallography, 2010. 66: p. 213-221.
61. Emsley, P., et al., Features and development of Coot. Acta Crystallographica Section D-Biological Crystallography, 2010. 66: p. 486-501.
62. Laskowski, R.A., et al., PROCHECK - a program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 1993. 26: p. 283-291.
63. Chen, V.B., et al., MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica Section D-Structural Biology, 2010. 66: p. 12-21.
64. Luscombe, N.M., R.A. Laskowski, and J.M. Thornton, NUCPLOT: a program to generate schematic diagrams of protein-nucleic acid interactions. Nucleic Acids Res, 1997. 25(24): p. 4940-5.
65. Kay, L.E., Pulsed field gradient multi-dimensional NMR methods for the study of protein structure and dynamics in solution. Prog Biophys Mol Biol, 1995. 63(3): p. 277-99.
66. Johnson, B.A. and R.A. Blevins, NMR VIEW - a computer-program for the visualization and analysis of nmr data. Journal of Biomolecular Nmr, 1994. 4(5): p. 603-614.
67. Keller, R.L.J., Optimizing the process of nuclear magnetic resonance spectrum analysis and computer aided resonance assignment. 2005.
68. Shen, Y., et al., Consistent blind protein structure generation from NMR chemical shift data. Proc Natl Acad Sci U S A, 2008. 105(12): p. 4685-90.
69. Shen, Y., et al., De novo protein structure generation from incomplete chemical shift assignments. J Biomol NMR, 2009. 43(2): p. 63-78.
70. Head, C.G., A. Tardy, and L.J. Kenney, Relative binding affinities of OmpR and OmpR-phosphate at the ompF and ompC regulatory sites. Journal of Molecular Biology, 1998. 281(5): p. 857-870.
71. Martinez-Hackert, E. and A.M. Stock, Structural relationships in the OmpR family of winged-helix transcription factors. J Mol Biol, 1997. 269(3): p. 301-12.
72. Blanco, A.G., et al., Tandem DNA recognition by PhoB, a two-component signal transduction transcriptional activator. Structure, 2002. 10(5): p. 701-13.
73. Yoshida, T., et al., Transcription regulation of ompF and ompC by a single transcription factor, OmpR. J Biol Chem, 2006. 281(25): p. 17114-23.
74. Pickard, D., et al., Characterization of defined OmpR mutants of Salmonella-typhi - OmpR is involved in the regulation of vi polysaccharide expression. Infection and Immunity, 1994. 62(9): p. 3984-3993.
75. Lewis, R.J., et al., Domain swapping in the sporulation response regulator SpoOA. Journal of Molecular Biology, 2000. 297(3): p. 757-770.