(3.238.130.97) 您好!臺灣時間:2021/05/14 19:43
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

: 
twitterline
研究生:游惟鈞
研究生(外文):Wei-Chun Yu
論文名稱:C-di-AMP在Bacillus subtilis中調控鉀離子轉運蛋白KtrAB的機制
論文名稱(外文):The regulation of KtrAB potassium transporter with c-di-AMP in Bacillus subtilis
指導教授:胡念仁
指導教授(外文):Nien-Jen Hu
口試委員:周三和馬徹
口試委員(外文):Shan-Ho ChouChe Ma
口試日期:2016-07-22
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:77
中文關鍵詞:鉀離子運轉蛋白
外文關鍵詞:C-di-AMPKtrAB
相關次數:
  • 被引用被引用:0
  • 點閱點閱:21
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
鉀離子(K+)是生物細胞內主要的陽離子,具有維持滲透壓、調節pH值、抵抗乾旱等重要的生理意義,因此調控K+運輸途徑的研究一直備受關注。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。在近期研究中發現,KtrA能與細菌中新型二級訊號分子cyclic diadenosine monophosphate (c-di-AMP)具有專一性的親和力,因此推測c-di-AMP可能會藉與KtrA octamer ring的結合,間接調控KtrB運輸K+的能力。然而,因為缺乏KtrAB complex與c-di-AMP的晶體結構證據,仍無法確認c-di-AMP調節KtrAB complex所使用的分子機制。在本研究中,我們利用分子篩層析(SEC)的方式確認缺乏核苷酸結合的KtrA依舊具有與KtrB形成KtrAB complex的能力。使用等溫滴定量熱法(ITC)法測量此種KtrA與c-di-AMP間有相當高的親和力。此外,我們分別以動態光散射法 (DLS)及分析級超高速離心機法(AUC),去檢測KtrA octamer ring在添加c-di-AMP後是否會有構型被改變或破壞的情況發生,結果發現KtrA octamer ring不會因此分離,但是ring的構形可能有所改變。我們也嘗試以nucleotide- less KtrA與c-di-AMP共結晶,並已取得初步的結晶條件。期望藉由完整的KtrA octamer ring與c-di-AMP晶體結構,能提供更深入了解c-di-AMP在調控K+運輸中所用機制的資訊。

Potassium ions (K+) are critical in bacterial cells with important physiological functions, such as osmotic homeostasis, pH regulation and resistance to draught. Therefore, regulation of potassium ion concentration has been a crucial question for scientists. Previous studies have shown that KtrAB complex, a member of Ktr transporter, exhibiting a vital role in resisting osmotic stress and high salinity by mediating uptake of K+ into cells. The crystal structure of the KtrAB complex from Bacillus subtilis shows that it is composed of a transmembrane potassium channel (KtrB) and a cytosolic regulatory protein (KtrA). In the complex structure KtrB assembles as homodimers while KtrA forms an octameric ring next to the KtrB dimer. The structural data also showed that KtrA adopts different conformations when binding to ATP or ADP. Recent biochemical studies showed that KtrA has a specific binding affinity with cyclic diadenosine monophosphate (c-di-AMP), a novel class of second messenger in bacteria. It is thus speculated that c-di-AMP might regulate K+ transportation of KtrB via the conformation change of KtrA signaled by this nucleotide molecule. So far the molecular mechanism of c-di-AMP in regulating the function of KtrAB complex remains unclear due to the lack of structural information of KtrAB in complex with c-di-AMP. Here we demonstrated that nucleotide-less KtrA and KtrB form complex in solution judged from the SEC profiles. We also used ITC to determine the specific affinity between this KtrA and c-di-AMP. Moreover, we further used DLS and AUC method to determine the KtrA octamer ring state in the presence of c-di-AMP. The result suggests that the ring remains its octameric state, but with some conformation changes. Cocrystallization of KtrA with c-di-AMP was attempted and preliminary hits were obtained. The crystal structure of KtraAB in complex with c-di-AMP will provide insightful information into how conformation changes of KtrA upon c-di-AMP binding modulates the K+ conductance of KtrB.

目次
中文摘要 i
Abstract ii
目次 iii
圖目次 vi
縮寫檢索表 viii
第一章 前言 1
一、 生物體中鉀離子的重要性 1
二、 K+-transporter proteins 1
三、 K+-transporter蛋白-KtrB 3
四、 胞質調控蛋白-KtrA 5
五、 新型二級訊號分子:c-di-AMP 7
六、 c-di-AMP近期研究 8
七、 本論文研究目的 10
第二章 材料與方法 12
一、 KtrA、KtrB質體建構 12
(一) 全長蛋白之DNA序列取得 12
(二) 引子設計 12
(三) 聚合酶連鎖反應(polymerase chain reaction, PCR) 14
(四) 以DNA膠體電泳 17
(五) 進行PCR product處理 17
(六) 重組DNA 18
(七) 製備勝任細胞 19
(八) 轉殖作用(transformation) 19
(九) 以Colony-PCR確認接合狀況 19
(十) 全長KtrA、KtrB基因定序 20
二、 KtrA蛋白之大量表現及純化 20
(一) 全長KtrA之小規模(small scale)表現條件篩選 20
(二) 大規模(large scale)表現 21
(三) 取得水溶性蛋白KtrA混合液 21
(四) 第一道純化管柱–離子交換樹脂(GigaCap® Q-650M column) 22
(五) 第二道純化管柱–Adenosine 5''-diphosphate-Agarose(ADP agarose, SIGMA) 22
(六) 透析排除ATP 22
(七) 第三道純化管柱–膠體過濾法(gel filtration) 23
(八) 蛋白質確認及取得 23
(九) 蛋白質濃度測定 24
三、 KtrB蛋白之大量表現及純化 24
(一) 全長KtrB之小規模(small scale)表現條件篩選 24
(二) 大規模(large scale)表現 25
(三) 取得膜蛋白KtrB混合液 25
(四) 第一道純化管柱–親和性管柱(Ni-NTA) 26
(五) 第二道純化管柱–His TrapTMHP column 26
(六) 第三道純化管柱–膠體過濾法(gel filtration) 27
(七) 蛋白質確認及取得 27
(八) 蛋白質濃度測定 28
四、 其他生物物理實驗方法 28
(一) 以Gel-filtration chromatography觀察KtrA/KtrB complex形成 28
(二) 測量法Isothermal Titration Calorimetry ( ITC) 29
(三) 測量法Dynamic Light Scattering (DLS) 29
(四) 測量法Analytical Ultracentrifugation (AUC) 30
五、 結晶條件篩選測試 30
(一) 結晶前置準備 30
(二) 結晶及觀測 31
第三章 結果 32
一、 純化來自Bacillus subtilis的KtrA和KtrB 32
(一) 進行KtrA純化 32
(二) 進行KtrB純化 33
二、 KtrA 在N_domain未結合ATP、ADP的狀況下可與KtrB形成KtrAB complex 34
三、 全長KtrA與c-di-AMP的親和力 35
四、 C-di-AMP對KtrA八聚體構型的影響 35
(一) 動態光散射法 (DLS) 36
(二) 分析級超高速離心機法(AUC) 36
五、 KtrA與c-di-AMP結晶測試 37
第四章 討論 38
一、 比較不同RCK C_domain之差異 38
二、 KtrA RCK C_domain結合c-di-AMP對KtrA 構型的影響 40
三、 ATP對KtrAB complex的影響 41
四、 探討KtrB能夠形成dimer的機制 43
五、 研究展望 46
參考文獻 47

圖目次
圖一、 KcsA topology 53
圖二、 KcsA結構圖 (Inga Hänelt, Tholema et al. 2011) 54
圖三、 KtrAB complex結構圖 (Vieira-Pires, Szollosi et al. 2013) 55
圖四、 KtrB Topology 56
圖五、 KtrB monomer結構圖 (Vieira-Pires, Szollosi et al. 2013) 57
圖六、 Trk 系統 58
圖七、 各種RCK調控蛋白結構圖 59
圖八、 KtrA octamer ring 結合ATP、ADP構型變化 60
圖九、 KtrAB complex間的Lateral-contact、Tip contacts親和力 61
圖十、 KtrA RCK domain與c-di-AMP反應位置 62
圖十一、 C-di-AMP與KtrASa C_domain dimer結合圖 63
圖十二、 CpaA RCK_CTD與c-di-AMP結構圖 (Ko-Hsin Chin, Liang et al. 2015) 64
圖十三、 C-di-AMP藉由結合Streptococcus pneumoniae CabP以調控鉀離子運輸蛋白SPD_0076示意圖 65
圖十四、 In-Fusion 構築質體所用之primer設計範例 66
圖十五、 KtrA-pET21 質體之構築 67
圖十六、 全長KtrA 純化SEC及SDS-PAGE結果圖 68
圖十七、 KtrB-DHHis vector 質體之構築 69
圖十八、 KtrB 純化SEC及SDS-PAGE結果圖 70
圖十九、 KtrAB complex形成之SEC profile及SDS-PAGE圖 71
圖二十、 以ITC測試透析ATP後之全長KtrA與c-di-AMP結果圖 72
圖二十一、 動態光散射法 (DLS)測量結果 73
圖二十二、 分析級超高速離心機法(AUC)測量結果 74
圖二十三、 全長KtrA與c-di-AMP結晶測試 75
圖二十四、 不同菌株KtrA與MthK RCK C_domain圖 (Vieira-Pires, Szollosi et al. 2013) (Henna Kim, Youn et al. 2015) (Hay Dvir, Valera et al. 2010) 76
圖二十五、 KtrB與TrkH結構圖 (Vieira-Pires, Szollosi et al. 2013) (Yu Cao, Pan et al. 2013) 77



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. Progress in Nucleic Acid Research and Molecular Biology, Vol 75, 2003. 75: p. 293-320.
4.Monro, R.E., Catalysis of peptide bond formation by 50 S ribosomal subunits from Escherichia coli. J Mol Biol, 1967. 26(1): p. 147-51.
5.Maden, B.E., R.R. Traut, and R.E. Monro, Ribosome-catalysed peptidyl transfer: the polyphenylalanine system. J Mol Biol, 1968. 35(2): p. 333-45.
6.Nissen, P., et al., The structural basis of ribosome activity in peptide bond synthesis. Science, 2000. 289(5481): p. 920-930.
7.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.
8.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. Biophysical Journal, 1999. 77(2): p. 789-807.
9.Durell, S.R., E.P. Bakker, and H.R. Guy, Does the KdpA subunit from the high affinity K+-translocating P-type KDP-ATPase have a structure similar to that of K+ channels? Biophysical Journal, 2000. 78(1): p. 188-199.
10.Schrempf, H., et al., A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans. EMBO J, 1995. 14(21): p. 5170-8.
11.Doyle, D.A., et al., The structure of the potassium channel: Molecular basis of K+ conduction and selectivity. Science, 1998. 280(5360): p. 69-77.
12.Jiang, Y.X., et al., Crystal structure and mechanism of a calcium-gated potassium channel. Nature, 2002. 417(6888): p. 515-522.
13.Kuo, A.L., et al., Crystal structure of the potassium channel KirBac1.1 in the closed state. Science, 2003. 300(5627): p. 1922-1926.
14.Miller, C., An overview of the potassium channel family. Genome Biol, 2000. 1(4): p. REVIEWS0004.
15.Durell, S.R., et al., Evolutionary relationship between K(+) channels and symporters. Biophysical Journal, 1999. 77(2): p. 775-788.
16.Liu, Y.S., P. Sompornpisut, and E. Perozo, Structure of the KcsA channel intracellular gate in the open state. Nat Struct Biol, 2001. 8(10): p. 883-7.
17.Hille, B., C.M. Armstrong, and R. MacKinnon, Ion channels: From idea to reality. Nature Medicine, 1999. 5(10): p. 1105-1109.
18.Zhou, Y.F., et al., Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 angstrom resolution. Nature, 2001. 414(6859): p. 43-48.
19.MacKinnon, R., Potassium channels and the atomic basis of selective ion conduction. Bioscience Reports, 2004. 24(2): p. 75-100.
20.Hirano, M., et al., Role of the KcsA Channel Cytoplasmic Domain in pH-Dependent Gating. Biophysical Journal, 2011. 101(9): p. 2157-2162.
21.Yuchi, Z.G., V.P.T. Pau, and D.S.C. Yang, GCN4 enhances the stability of the pore domain of potassium channel KcsA. Febs Journal, 2008. 275(24): p. 6228-6236.
22.Bhate, M.P. and A.E. McDermott, Protonation state of E71 in KcsA and its role for channel collapse and inactivation. Proceedings of the National Academy of Sciences of the United States of America, 2012. 109(38): p. 15265-15270.
23.Uozumi, N., et al., The Arabidopsis HKT1 gene homolog mediates inward Na+ currents in Xenopus laevis oocytes and Na+ uptake in Saccharomyces cerevisiae. Plant Physiology, 2000. 122(4): p. 1249-1259.
24.Horie, T., et al., Two types of HKT transporters with different properties of Na+ and K+ transport in Oryza sativa. Plant Journal, 2001. 27(2): p. 129-138.
25.Maser, P., et al., Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants. Proceedings of the National Academy of Sciences of the United States of America, 2002. 99(9): p. 6428-6433.
26.Hesse, J.E., et al., Sequence Homology between 2 Membrane-Transport Atpases, the Kdp-Atpase of Escherichia-Coli and the Ca-2+-Atpase of Sarcoplasmic-Reticulum. Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences, 1984. 81(15): p. 4746-4750.
27.Dong, J.B., et al., Structures of the MthK RCK domain and the effect of Ca2+ on gating ring stability. Journal of Biological Chemistry, 2005. 280(50): p. 41716-41724.
28.Bossemeyer, D., et al., K+-Transport Protein Trka of Escherichia-Coli Is a Peripheral Membrane-Protein That Requires Other Trk Gene-Products for Attachment to the Cytoplasmic Membrane. Journal of Biological Chemistry, 1989. 264(28): p. 16403-16410.
29.Cao, Y., et al., Gating of the TrkH ion channel by its associated RCK protein TrkA. Nature, 2013. 496(7445): p. 317-+.
30.Nakamura, T., et al., KtrAB, a new type of bacterial K+-uptake system from Vibrio alginolyticus. Journal of Bacteriology, 1998. 180(13): p. 3491-3494.
31.Holtmann, G., et al., KtrAB and KtrCD: Two K+ uptake systems in Bacillus subtilis and their role in adaptation to hypertonicity. Journal of Bacteriology, 2003. 185(4): p. 1289-1298.
32.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.
33.Tholema, N., et al., All four putative selectivity filter glycine residues in KtrB are essential for high affinity and selective K+ uptake by the KtrAB system from Vibrio alginolyticus. Journal of Biological Chemistry, 2005. 280(50): p. 41146-41154.
34.Albright, R.A., K. Joh, and J.H. Morais-Cabral, Probing the structure of the dimeric KtrB membrane protein. Journal of Biological Chemistry, 2007. 282(48): p. 35046-35055.
35.Hanelt, I., et al., KtrB, a member of the superfamily of K+ transporters. European Journal of Cell Biology, 2011. 90(9): p. 696-704.
36.Cordero-Morales, J.F., et al., Molecular determinants of gating at the potassium-channel selectivity filter. Nature Structural & Molecular Biology, 2006. 13(4): p. 311-318.
37.Uysal, S., et al., Mechanism of activation gating in the full-length KcsA K+ channel. Proceedings of the National Academy of Sciences of the United States of America, 2011. 108(29): p. 11896-11899.
38.Cao, Y., et al., Crystal structure of a potassium ion transporter, TrkH. Nature, 2011. 471(7338): p. 336-40.
39.Levin, E.J. and M. Zhou, Recent progress on the structure and function of the TrkH/KtrB ion channel. Current Opinion in Structural Biology, 2014. 27: p. 95-101.
40.Albright, R.A., et al., The RCK domain of the KtrAB K+ transporter: Multiple conformations of an octameric ring. Cell, 2006. 126(6): p. 1147-1159.
41.Szollosi, A., et al., Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter. Plos Biology, 2016. 14(1).
42.Pau, V.P., et al., Crystal Structure and Asymmetric Conformation of a K+ Channel RCK Domain. Biophysical Journal, 2016. 110(3): p. 291a-291a.
43.Smith, F.J., et al., Multiple Ca2+ Binding Sites in the RCK Domain Contribute to Gating in the MthK K+ Channel. Biophysical Journal, 2012. 102(3): p. 690a-690a.
44.Zhao, T.Z., et al., Molecular Dynamics Simulations of Calcium Binding Sites in the RCK Domain of the Mthk Gating Ring. Biophysical Journal, 2015. 108(2): p. 435a-435a.
45.Pico, A.R. and R. MacKinnon, Role of the RCK domain in BK channel gating. Biophysical Journal, 2002. 82(1): p. 228a-229a.
46.Krishnamoorthy, G., J.Y. Shi, and J.M. Cui, Structural motifs in the RCK domain modulate Ca2+ sensitivity of BK channel activation. Biophysical Journal, 2003. 84(2): p. 7a-8a.
47.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. Journal of Biological Chemistry, 2007. 282(19): p. 14018-14027.
48.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.
49.Liu, Y., et al., Gated access to the pore of a voltage-dependent K+ channel. Neuron, 1997. 19(1): p. 175-184.
50.Benveniste, E.N., et al., Second messenger systems in the regulation of cytokines and adhesion molecules in the central nervous system. Brain Behavior and Immunity, 1995. 9(4): p. 304-314.
51.Stahl, S.M., Second messenger systems. Psychiatric Annals, 1996. 26(4): p. 183-184.
52.Sorensen, P.W. and K. Sato, Second messenger systems mediating sex pheromone and amino acid sensitivity in goldfish olfactory receptor neurons. Chemical Senses, 2005. 30: p. I315-I316.
53.Steiner, A.A., J. Antunes-Rodrigues, and L.G.S. Branco, Role of preoptic second messenger systems (cAMP and cGMP) in the febrile response. Brain Research, 2002. 944(1-2): p. 135-145.
54.Romling, U., M. Gomelsky, and M.Y. Galperin, C-di-GMP: the dawning of a novel bacterial signalling system. Molecular Microbiology, 2005. 57(3): p. 629-639.
55.Romling, U., Great Times for Small Molecules: c-di-AMP, a Second Messenger Candidate in Bacteria and Archaea. Science Signaling, 2008. 1(33).
56.Boyd, C.D. and G.A. O''Toole, Second Messenger Regulation of Biofilm Formation: Breakthroughs in Understanding c-di-GMP Effector Systems. Annual Review of Cell and Developmental Biology, Vol 28, 2012. 28: p. 439-462.
57.Burdette, D.L., et al., STING is a direct innate immune sensor of cyclic di-GMP. Nature, 2011. 478(7370): p. 515-U111.
58.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.
59.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. 1703-1705.
60.Rao, F., et al., YybT Is a Signaling Protein That Contains a Cyclic Dinucleotide Phosphodiesterase Domain and a GGDEF Domain with ATPase Activity. Journal of Biological Chemistry, 2010. 285(1): p. 473-482.
61.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).
62.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 (Amst), 2015. 27: p. 1-8.
63.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.
64.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.
65.Zhang, L., W.H. Li, and Z.G. He, DarR, a TetR-like Transcriptional Factor, Is a Cyclic Di-AMP-responsive Repressor in Mycobacterium smegmatis. Journal of Biological Chemistry, 2013. 288(5): p. 3085-3096.
66.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.
67.Oppenheimer-Shaanan, Y., et al., c-di-AMP reports DNA integrity during sporulation in Bacillus subtilis. Embo Reports, 2011. 12(6): p. 594-601.
68.Bai, Y., et al., Cyclic di-AMP impairs potassium uptake mediated by a cyclic di-AMP binding protein in Streptococcus pneumoniae. J Bacteriol, 2014. 196(3): p. 614-23.
69.Chin, K.H., et al., Structural Insights into the Distinct Binding Mode of Cyclic Di-AMP with SaCpaA_RCK. Biochemistry, 2015. 54(31): p. 4936-4951.
70.Szollosi, A., et al., Dissecting the Molecular Mechanism of Nucleotide-Dependent Activation of the KtrAB K+ Transporter. PLoS Biol, 2016. 14(1): p. e1002356.
71.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.



QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top