臺灣博碩士論文加值系統

在細菌中鉀離子(K+)具有維持滲透壓、調節 pH 質、抵抗乾旱等重要的生理功 能。過去研究顯示，屬於 potassium ion transporters 一員的 KtrAB complex 在許多 細菌中扮演了調控 K+的關鍵角色，能藉由調節吸收 K+來抵抗高滲透壓、高鹽環 境以維持細菌穩定生存。透過解析 Bacillus subtilis KtrAB complex 的晶體學結構， 已知 KtrAB complex 是由二聚體的跨膜運輸蛋白 KtrB 以及八聚體環的細胞質蛋白 KtrA 所構成，而且 KtrA octamer ring 在結合 ATP 或是 ADP 會呈現不同的構型， 進而調控 KtrB 運輸 K+的能力。KtrA 屬於 Regulator of Conductance of K+ (RCK)家 族，其結構包含 RCK_N domain 和 RCK_C domain 所組成。RCK_N domain 已被證 實含有 ATP 和 ADP 的結合位，而 RCK_C domain 則含有新型二級訊號分子 cyclic diadenosine monophosphate (c-di-AMP)結合位，因此推測 c-di-AMP 可能會藉由與 KtrA octamer ring 的結合，間接調控 KtrB 運輸 K+的能力。雖然在過去研究已得到 RCK_C domain 和 c-di-AMP 晶體結構，但在缺乏全長 KtrA 與 c-di-AMP 的晶體結 構的輔助，仍無法確認 c-di-AMP 調節 KtrAB complex 所使用的分子機制。因此我 們純化了全長 KtrA，並使用 ITC 測定全長 KtrA 和 c-di-AMP 之間的親和力為 Kd ∼430nM。 接著我們共結晶全長 KtrA 和 c-di-AMP，並收集完整的 X-ray 衍射數 據，其解析度高達 2.8A，並以 Molecular Replacement 得到結構。然而，在我們的 結構中發現 ATP 結合在 RCK_N domain，而在 RCK_C domain 並沒有看到 c-di- AMP。將此解出結構與過去研究相比較，發現 ATP 的結合改變了 RCK_C domain 的構型，因此抑制 c-di-AMP 的結合。接著以電子顯微鏡及蛋白交聯等實驗，觀察 到 c-di-AMP 可能會抑制 KtrA 八聚體環的形成。我們因此提出假說:c-di-AMP 結 合 KtrAB complex 時會造成 KtrA 與 KtrB 解離，進而抑制 K+運輸。

In bacterial cells, KtrAB complex is critical in resistance to osmotic stress by mediating uptake of K+ into cell. The crystal structure of KtrAB from Bacillus subtilis shows that KtrAB complex is composed of a dimeric potassium channel KtrB and a cytoplasmic protein KtrA forming an octameric ring beneath KtrB. Both structural and biochemical studies suggested that nucleotide-binding, such as ATP and ADP, to the KtrA octamer ring changes the conformation of the ring, and thus regulates the K+ transportation of KtrB. KtrA is a member of Regulator of Conductance of K+ (RCK) family and contains RCK_N and RCK_C domains. It has been demonstrated that RCK_N domain harbors the binding sites for ATP and ADP, and RCK_C domain has a specific affinity with c-di-AMP, a novel second messenger. The co-crystal structure of KtrA RCK_C domain with c-di-AMP was solved previously; however, the complex structure of full-length (FL) KtrA with c-di-AMP is still undetermined. As a result, how c-di-AMP binding evokes conformational change of KtrA and, subsequently, mediates the transport activity of KtrB, remains illusive. Here we purified FL-KtrA and then determined the affinity between FL-KtrA and c-di-AMP to be Kd ~ 430 nM by using ITC. We co- crystallized FL-KtrA with c-di-AMP, and a complete X-ray diffraction dataset was collected. The structure was solved by Molecular Replacement. However, two ATPs were observed at the RCK_N domain in our structure, and no c-di-AMP near the RCK_C dimer interface was localized. Comparing the structure with previous studies, we found that the binding of ATP would change the conformation of RCK_C domain and thus inhibit c-di- AMP binding. We also found c-di-AMP would inhibit KtrA forming octamer ring by using electron microscope and crosslinking. We hypothesized that the binding of c-di- AMP will induce KtrA and KtrB dissociations and thus inhibit K+ transportation.

中文摘要 ............................................. i
Abstract ........................................... ii
目次 ....................................iii
圖目次 ............................................ vi
第一章 前言 .................................1
ㄧ、鉀離子的重要性 ................................... 1
二、鉀離子通道蛋白 ...............................1
(一) KcsA 鉀離子通道蛋白 .........................1
(二) RCK (Regulator of Conductance of K+) domain....3
三、 KtrAB complex.......................................3
(一) 鉀離子通道蛋白-KtrB..............................3
(二) 細胞質調控蛋白-KtrA...........................4
四、新型二級訊號分子 c-di-AMP ....................... 5
五、KtrA 與 c-di-AMP 的近期研究 ..................... 6
六、本論文研究目的 .............................7
第二章 材料與方法 ............................. 9
一、Tagless KtrA 蛋白之大量表現及純化..............9
(一) Tagless KtrA 蛋白大規模(large scale)表現 ....... 9
(二) 取得水溶性蛋白 tagless KtrA 混合液 ...................9
(三) 第一道純化管柱–離子交換樹脂(GigaCapR Q-650M column).........................9
(四) 第二道純化管柱– ADP agarose ................. 10
(五) 透析排除 ATP.............................10
(六) 第三道純化管柱–膠體過濾法(gel filtration).....10
(七) 蛋白質確認及取得...............................11
(八) 蛋白質濃度測定....................11
二、His12-KtrA 蛋白之大量表現及純化.............11
(一) His12-KtrA 蛋白大規模(large scale)表現 ........11
(二) 取得水溶性蛋白His12-KtrA混合液......................12
(三) 第一道純化管柱–親和性管柱(Ni-NTA)...........12
(四) 第二道純化管柱–His TrapTMHP column .........12
iii
(五) 第三道純化管柱–膠體過濾法(gel filtration).......13
(六) 蛋白質確認及取得.............................13
(七) 蛋白質濃度測定...............................13
三、KtrA-His6 質體建構、蛋白大量表現及純化 ...........14
(一) KtrA-His6 質體建構 ...........................14
(二) KtrA-His6 蛋白大規模(large scale)表現.........15
(三) 取得水溶性蛋白KtrA-His6混合液..............16
(四) 第一道純化管柱–親和性管柱(Ni-NTA).............16
(五) 第二道純化管柱–膠體過濾法(gel filtration)....16
(六) 蛋白質確認及取得...............................16
(七) 蛋白質濃度測定.............................16
四、KtrA-His6 I78V 質體建構、蛋白大量表現及純化....17
(一) KtrA-His6 I78V 質體建構......................17
(二) KtrA-His6 I78V 大規模(large scale)表現及純化.. 18
五、KtrB 蛋白之大量表現及純化 ....................18
(一) 大規模(large scale)表現......................18
(二) 取得膜蛋白 KtrB solubilized 混合液..........18
(三) 第一道純化管柱–親和性管柱(Ni-NTA)...........19
(四) 第二道純化管柱–His TrapTMHP column ...........19
(五) 第三道純化管柱–膠體過濾法(gel filtration).....19
(六) 蛋白質確認及取得................................20
(七) 蛋白質濃度測定................................20
六、VcDncV 純化及 c-di-AMP 合成 ..........20
(一) 大規模(large scale)表現...........................20
(二) 取得水溶性蛋白VcDncV混合液................21
(三) 純化管柱–親和性管柱(Ni-NTA)...............21
(四) 蛋白質確認及取得...............................21
(五) c-di-AMP 合成 .................................. 21
(六) c-di-AMP 純化- Reverse phase chromatography.....22
(七) c-di-AMP 濃度計算 .......................... 22
七、KtrAB complex 純化............................22
八、結晶條件篩選測試 ............................23
(一) KtrA tag less、KtrA-His6 和 KtrAB complex 結晶前置準備........................... 23
(二) 點晶及晶體觀察.......................23
(三)taglessKtrA 結晶條件微調....................23
九、Isothermal Titration Calorimetry (ITC).....24
十、X-ray cystalography..........................24
iv
十一、穿透式電子顯微鏡(Electron Microscope) ............. 24
十二、蛋白交聯(Crosslinking) .................25
十三、KtrA-His6 和 KtrA-His6 I78V 蛋白表現條件篩選..25
第三章 結果 ...................................27
一、Tagless KtrA 與 c-di-AMP 有高度親和力............27
(一) tagless KtrA 純化 ..........................27
(二) VcDncV 純化及 c-di-AMP 合成 .............27
(三) 等溫滴定量熱法(Isothermal Titration Calorimetry, ITC)................................28
二、c-di-AMP 會影響 tagless KtrA 八聚體環的形成 ..28
(一) 電子顯微鏡(ElectronMicroscope)..................28
(二) 蛋白交聯(Crosslinking).......................28
三、Tagless KtrA 可與 KtrB 形成 KtrAB complex ... 29
(一) KtrB 純化 ................................... 29
(二) KtrAB complex 純化............................30
(三) KtrAB complex 與 c-di-AMP 共結晶 .......... 30
四、tagless KtrA 與 c-di-AMP 共結晶試驗 ............31
(一) 共結晶 tagless KtrA 與 c-di-AMP ................ 31
(二) tagless KtrA 與 c-di-AMP 晶體結構 ..........31
五、His12-KtrA 純化................................32
六、KtrA-His6 質體建構與純化 ....................33
(一) pET21a-KtrA-His6 質體建構 ..................33
(二) KtrA-His6 蛋白純化 ..........................33
七、KtrA-His6 I78V 質體建構與純化.................34
(一)pET21a-KtrA-His6-I78V 點突變.................34
(二) KtrA-His6 I78V 純化............................34
八、KtrA-His6 和 c-di-AMP 有高度親和力 ..........35
九、KtrA-His6 失去結合 ATP 的能力...................35
十、共結晶 KtrA-His6 與 c-di-AMP ...............35
十一、KtrA-His6 及 KtrA-His6 I78V 蛋白表現條件篩選 ..36
第四章、討論 ...................................... 37
一、KtrA 調控機制-ATP 異位調控 c-di-AMP ..........37
二、c-di-AMP 與 KtrAB complex 的調控機制探討 ...... 38
三、比較不同 RCK domain 之差異 .................... 38
四、KtrA 與 c-di-AMP 共結晶的瓶頸 ................ 39
五、未來研究展望 .................................39
參考文獻 ........................................... 41

1. Booth, I.R., Regulation of cytoplasmic pH in bacteria. Microbiol Rev, 1985. 49(4): p. 359-78.
2. Bakker, E.P., Alkali cation transport systems in prokaryotes. 1993, Boca Raton: CRC Press. 448 p.
3. Epstein, W., The roles and regulation of potassium in bacteria. Prog Nucleic Acid Res Mol Biol, 2003. 75: p. 293-320.
4. Miller, C., An overview of the potassium channel family. Genome Biol, 2000. 1(4): p. REVIEWS0004.
5. Littleton, J.T. and B. Ganetzky, Ion channels and synaptic organization: analysis of the Drosophila genome. Neuron, 2000. 26(1): p. 35-43.
6. Wojdan, A., M.E. Morin, and G. Oshiro, Effects of Potassium Channel Activators on Blood-Pressure and Heart-Rate in Spontaneously Hypertensive Rats after Oral and Intravenous Treatment. Faseb Journal, 1988. 2(4): p. A607-A607.
7. Meredith LeMasurier, L.H., and Christopher Miller, KcsA: It’s a Potassium Channel. J. Gen. Physiol., 2001. 118.
8. Declan A. Doyle, J.o.M.C., Richard A. Pfuetzner, Anling Kuo, Jacqueline M. Gulbis, Steven L. Cohen, Brian T. Chait, Roderick MacKinnon*, The Structure of the Potassium Channel: Molecular Basis of K Conduction and Selectivity. Science, 1998. 280.
9. Kuo, A., et al., Crystal structure of the potassium channel KirBac1.1 in the closed state. Science, 2003. 300(5627): p. 1922-6.
10. Youxing Jiang, A.L., Jiayun Chen, Martine Cadene, Brian T. Chait & Roderick MacKinnon, Crystal structure and mechanism of a calcium-gated potassium channel. Nature, 2002. 417.
11. Vieira-Pires, R.S., A. Szollosi, and J.H. Morais-Cabral, The structure of the KtrAB potassium transporter. Nature, 2013. 496(7445): p. 323-8.
12. Cao, Y., et al., Gating of the TrkH ion channel by its associated RCK protein TrkA. Nature, 2013. 496(7445): p. 317-22.
13. Albright, R.A., et al., The RCK domain of the KtrAB K+ transporter: multiple conformations of an octameric ring. Cell, 2006. 126(6): p. 1147-59.
14. Cuello, L.G., et al., pH-dependent gating in the Streptomyces lividans K+ channel. Biochemistry, 1998. 37(10): p. 3229-36.
41
15. Deisenhofer, J., et al., Structure of the protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3A resolution. Nature, 1985. 318(6047): p. 618-24.
16. Hanelt, I., et al., KtrB, a member of the superfamily of K+ transporters. Eur J Cell Biol, 2011. 90(9): p. 696-704.
17. Zhou, Y., et al., Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution. Nature, 2001. 414(6859): p. 43-8.
18. MacKinnon, R., Potassium channels and the atomic basis of selective ion conduction (Nobel Lecture). Angew Chem Int Ed Engl, 2004. 43(33): p. 4265-77.
19. Yuchi, Z., V.P. Pau, and D.S. Yang, GCN4 enhances the stability of the pore
domain of potassium channel KcsA. FEBS J, 2008. 275(24): p. 6228-36.
20. Hirano, M., et al., Role of the KcsA channel cytoplasmic domain in pH-dependent
gating. Biophys J, 2011. 101(9): p. 2157-62.
21. Bhate, M.P. and A.E. McDermott, Protonation state of E71 in KcsA and its role
for channel collapse and inactivation. Proc Natl Acad Sci U S A, 2012. 109(38):
p. 15265-70.
22. Durell, S.R. and H.R. Guy, Structural models of the KtrB, TrkH, and Trk1,2
symporters based on the structure of the KcsA K(+) channel. Biophys J, 1999.
77(2): p. 789-807.
23. Youxing Jiang, A.L., Jiayun Chen, Martine Cadene, Brian T. Chait & Roderick
MacKinnon, The open pore conformation of potassium channels. Nature, 2002.
30.
24. Ye, S., et al., Crystal structures of a ligand-free MthK gating ring: Insights into
the ligand gating mechanism of K+ channels. Cell, 2006. 126(6): p. 1161-1173.
25. Cao, Y., et al., Crystal structure of a potassium ion transporter, TrkH. Nature,
2011. 471(7338): p. 336-40.
26. Wu, Y., et al., Structure of the gating ring from the human large-conductance
Ca(2+)-gated K(+) channel. Nature, 2010. 466(7304): p. 393-7.
27. Yuan, P., et al., Structure of the human BK channel Ca2+-activation apparatus at
3.0 A resolution. Science, 2010. 329(5988): p. 182-6.
28. Pau, V.P.T., et al., Structure and function of multiple Ca2+-binding sites in a K+
channel regulator of K+ conductance (RCK) domain. Proceedings of the National Academy of Sciences of the United States of America, 2011. 108(43): p. 17684- 17689.
29. Kuo, M.M., et al., Prokaryotic K(+) channels: from crystal structures to diversity. FEMS Microbiol Rev, 2005. 29(5): p. 961-85.
42
30. Loukin, S.H., et al., Microbial K+ channels. J Gen Physiol, 2005. 125(6): p. 521- 7.
31. Holtmann, G., et al., KtrAB and KtrCD: two K+ uptake systems in Bacillus subtilis and their role in adaptation to hypertonicity. J Bacteriol, 2003. 185(4): p. 1289- 98.
32. Hanelt, I., et al., Gain of function mutations in membrane region M2C2 of KtrB open a gate controlling K+ transport by the KtrAB system from Vibrio alginolyticus. J Biol Chem, 2010. 285(14): p. 10318-27.
33. Albright, R.A., K. Joh, and J.H. Morais-Cabral, Probing the structure of the dimeric KtrB membrane protein. J Biol Chem, 2007. 282(48): p. 35046-55.
34. Szollosi, A., et al., Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter. PLoS Biol, 2016. 14(1): p. e1002356.
35. Kroning, N., et al., ATP binding to the KTN/RCK subunit KtrA from the K+ - uptake system KtrAB of Vibrio alginolyticus: its role in the formation of the KtrAB complex and its requirement in vivo. J Biol Chem, 2007. 282(19): p. 14018-27.
36. Kim, H., et al., Structural Studies of Potassium Transport Protein KtrA Regulator of Conductance of K+ (RCK) C Domain in Complex with Cyclic Diadenosine Monophosphate (c-di-AMP). J Biol Chem, 2015. 290(26): p. 16393-402.
37. Nakamura, T., et al., KtrAB, a new type of bacterial K(+)-uptake system from Vibrio alginolyticus. J Bacteriol, 1998. 180(13): p. 3491-4.
38. Sorensen, P.W. and K. Sato, Second messenger systems mediating sex pheromone and amino acid sensitivity in goldfish olfactory receptor neurons. Chem Senses, 2005. 30 Suppl 1: p. i315-6.
39. Benveniste, E.N., et al., Second messenger systems in the regulation of cytokines and adhesion molecules in the central nervous system. Brain Behav Immun, 1995. 9(4): p. 304-14.
40. Hengge, R., Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol, 2009. 7(4): p. 263-73.
41. Romling, U., Great times for small molecules: c-di-AMP, a second messenger candidate in Bacteria and Archaea. Sci Signal, 2008. 1(33): p. pe39.
42. Burdette, D.L., et al., STING is a direct innate immune sensor of cyclic di-GMP. Nature, 2011. 478(7370): p. 515-U111.
43. Luo, Y. and J.D. Helmann, Analysis of the role of Bacillus subtilis sigma(M) in beta-lactam resistance reveals an essential role for c-di-AMP in peptidoglycan homeostasis. Mol Microbiol, 2012. 83(3): p. 623-39.
43
44. Zhang, L., W. Li, and Z.G. He, DarR, a TetR-like transcriptional factor, is a cyclic di-AMP-responsive repressor in Mycobacterium smegmatis. J Biol Chem, 2013. 288(5): p. 3085-96.
45. Oppenheimer-Shaanan, Y., et al., c-di-AMP reports DNA integrity during sporulation in Bacillus subtilis. EMBO Rep, 2011. 12(6): p. 594-601.
46. Bejerano-Sagie, M., et al., A checkpoint protein that scans the chromosome for damage at the start of sporulation in Bacillus subtilis. Cell, 2006. 125(4): p. 679- 690.
47. Corrigan, R.M., et al., Systematic identification of conserved bacterial c-di-AMP receptor proteins. Proc Natl Acad Sci U S A, 2013. 110(22): p. 9084-9.
48. Witte, G., et al., Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates. Molecular Cell, 2008. 30(2): p. 167-178.
49. Woodward, J.J., A.T. Iavarone, and D.A. Portnoy, c-di-AMP secreted by intracellular Listeria monocytogenes activates a host type I interferon response. Science, 2010. 328(5986): p. 1703-5.
50. Gandara, C. and J.C. Alonso, DisA and c-di-AMP act at the intersection between DNA-damage response and stress homeostasis in exponentially growing Bacillus subtilis cells. DNA Repair, 2015. 27: p. 1-8.
51. Yinlan Bai, J.Y., Tiffany M. Zarrella, Yang Zhang, Dennis W. Metzger, Guangchun Bai, Cyclic Di-AMP Impairs Potassium Uptake Mediated by a Cyclic Di-AMP Binding Protein in Streptococcus pneumoniae. Journal of Bacteriology, 2014. 96.
52. Santos, J.o.M.A., Assessment of the Binding Properties of KtrA to ATP and ADP. 2014.
53. Corrigan, R.M. and A. Grundling, Cyclic di-AMP: another second messenger enters the fray. Nat Rev Microbiol, 2013. 11(8): p. 513-24.
54. Davies, B.W., et al., Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for V. cholerae virulence. Cell, 2012. 149(2): p. 358-70.
55. Zhu, D., et al., Structural biochemistry of a Vibrio cholerae dinucleotide cyclase reveals cyclase activity regulation by folates. Mol Cell, 2014. 55(6): p. 931-7.
56. Corrigan, R.M., et al., c-di-AMP Is a New Second Messenger in Staphylococcus aureus with a Role in Controlling Cell Size and Envelope Stress. Plos Pathogens, 2011. 7(9).
57. Rosano, G.L. and E.A. Ceccarelli, Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol, 2014. 5: p. 172.
44
58. Smith, F.J., et al., Structural basis of allosteric interactions among Ca2+-binding sites in a K+ channel RCK domain. Nat Commun, 2013. 4: p. 2621.
59. Choe, S., Potassium channel structures. Nat Rev Neurosci, 2002. 3(2): p. 115-21.
60. Pau, V.P.T., K. Abarca-Heidemann, and B.S. Rothberg, Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel. Journal of
General Physiology, 2010. 135(5): p. 509-526.