二价金属离子与YycFN相互作用的NMR研究
收稿日期: 2015-03-30
修回日期: 2016-01-22
网络出版日期: 2016-03-05
基金资助
国家自然科学基金资助项目(21173257).
Divalent Metal Ion Binding to the Response Regulator YycFN Studied by NMR Spectroscopy
Received date: 2015-03-30
Revised date: 2016-01-22
Online published: 2016-03-05
YycGF最早发现于枯草芽孢杆菌,是与细胞存活密切相关的双组分信号转导系统,存在于少量低鸟嘌呤和胞嘧啶(G+C)含量的革兰氏阳性菌中,包括金黄色葡萄球菌和肺炎链球菌等人类病原菌.在外界环境刺激下,胞膜上的组氨酸激酶YycG通过自身组氨酸磷酸化活化,将磷酸根转移至反应调节蛋白YycF的N端调节区(YycFN)使之磷酸化,调控下游基因表达,实现特定细胞应答反应.二价金属离子在双组分信号转导系统反应调节蛋白的磷酸化过程中起着非常关键的作用,但它们与YycFN相互作用的机制尚不清楚.该文利用液体核磁共振(NMR)方法研究了Ca2+、Mg2+两种离子与YycFN的相互作用,对详细的相互作用界面进行了分析,并计算了Ca2+、Mg2+与YycFN的解离常数(Kd).发现金属离子的关键作用位点是Asp9、Asp16和Asp53等残基,蛋白的整体构象也发生了一定变化,为阐明二价金属离子在反应调节蛋白信号转导过程中的作用机制提供了重要线索.
刘婷 , 刘买利 , 姜凌 . 二价金属离子与YycFN相互作用的NMR研究[J]. 波谱学杂志, 2016 , 33(1) : 77 -88 . DOI: 10.11938/cjmr20160107
YycGF, originally identified in Bacillus subtilis, is recognized as a crucial two-component signal transduction system closely associated with cell viability. It is highly conserved in low G+C Gram-positive bacteria, including Staphylococcus aureus, Streptococcus pneumoniae and other human pathogens. The histidine kinase (HK) YycG senses extracellular or intracellular signals and phosphorylates its cognate response regulator (RR) YycF, which in turn recognizes sequence specific regions on the bacterial chromosome and regulates the expression of certain genes. The presence of a divalent metal ion is essential for phosphoryl group transfer. Here, we presented a metal ion binding study of the response regulator YycF (YycFN) from Bacillus subtilis using NMR spectroscopy. The metal ions Ca2+ and Mg2+ induced severe chemical shift changes in YycF backbone resonances, involving mainly the Asp9, Asp16 and Asp53 residues. Furthermore, the binding affinities of Ca2+ and Mg2+ with YycFN were compared. The results provide important clues for understanding the conformational change of YycFN upon metal ion binding before its phosphorylation.
Key words： NMR; metal ions; interaction; YycFN
[1] Stock A M, Robinson V L, Goudreau P N. Two-component signal transduction[J]. Annu Rev Biochem, 2000, 69: 183－215.
[2] Galperin M Y. Structural classification of bacterial response regulators: Diversity of output domains and domain combinations[J]. J Bacteriol, 2006, 188(12): 4 169－4 182.
[3] Fabret C, Feher V A, Hoch J A. Two-component signal transduction in Bacillus subtilis: How one organism sees its world[J]. J Bacteriol, 1999, 181(7): 1 975－1 983.
[4] Bourret R B. Receiver domain structure and function in response regulator proteins[J]. Curr Opin Microbiol, 2010, 13(2): 142－149.
[5] Lukat G S, Stock A M, Stock J B. Divalent metal ion binding to the CheY protein and its significance to phosphotransfer in bacterial chemotaxis[J]. Biochemistry, 1990, 29(23): 5 436－5 442.
[6] Needham J V, Chen T Y, Falke J J. Novel ion specificity of a carboxylate cluster Mg(II) binding site: strong charge selectivity and weak size selectivity[J]. Biochemistry, 1993, 32(13): 3 363－3 367.
[7] Hubbard J A, MacLachlan L K, King G W, et al. Nuclear magnetic resonance spectroscopy reveals the functional state of the signalling protein CheY in vivo in Escherichia coli[J]. Mol Microbiol, 2003, 49(5): 1 191－1 200.
[8] Kojetin D J, Thompson R J, Benson L M, et al. Structural analysis of divalent metals binding to the Bacillus subtilis response regulator Spo0F: the possibility for in vitro metalloregulation in the initiation of sporulation[J]. Biometals, 2005, 18(5): 449－466.
[9] Thompson J D, Higgins D G, Gibson T J. Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[J]. Nucleic Acids Res, 1994, 22(22): 4 673－4 680.
[10] Bisicchia P, Noone D, Lioliou E, et al. The essential YycFG two-component system controls cell wall metabolism in Bacillus subtilis[J]. Mol Microbiol, 2007, 65(1): 180－200.
[11] Fukushima T, Szurmant H, Kim E J, et al. A sensor histidine kinase co-ordinates cell wall architecture with cell division in Bacillus subtilis[J]. Mol Microbiol, 2008, 69(3): 621－632.
[12] Ahn S J, Burne R A. Effects of oxygen on biofilm formation and the AtlA autolysin of Streptococcus mutans[J]. J Bacteriol, 2007, 189(17): 6 293－6 302.
[13] Hancock L, Perego M. Two-component signal transduction in Enterococcus faecalis[J]. J Bacteriol, 2002, 184(21): 5 819－5 825.
[14] Dubrac S, Boneca I G, Poupel O, et al. New insights into the WalK/WalR (YycG/YycF) essential signal transduction pathway reveal a major role in controlling cell wall metabolism and biofilm formation in Staphylococcus aureus[J]. J Bacteriol, 2007, 189(22): 8 257－8 269.
[15] Dubrac S, Msadek T. Identification of genes controlled by the essential YycG/YycF two-component system of Staphylococcus aureus[J]. J Bacteriol, 2004, 186(4): 1 175－1 181.
[16] Howell A, Dubrac S, Andersen K K, et al. Genes controlled by the essential YycG/YycF two-component system of Bacillus subtilis revealed through a novel hybrid regulator approach[J]. Mol Microbiol, 2003, 49(6): 1 639－1 655.
[17] Watanabe T, Okada A, Gotoh Y, et al. Inhibitors targeting two-component signal transduction[J]. Adv Exp Med Biol, 2008, 631: 229－236.
[18] Qin Z Q, Zhang J, Xu B, et al. Structure-based discovery of inhibitors of the YycG histidine kinase: New chemical leads to combat Staphylococcus epidermidis infections[J]. BMC Microbiol, 2006, 6: 96－113.
[19] Schnell R, Agren D, Schneider G. 1.9 Å structure of the signal receiver domain of the putative response regulator NarL from Mycobacterium tuberculosis[J]. Acta Crystallogr F, 2008, 64(12): 1 096－1 100.
[20] Gao R, Mack T R, Stock A M. Bacterial response regulators: Versatile regulatory strategies from common domains[J]. Trends Biochem Sci, 2007, 32(5): 225－234.
[21] Lukat G S, McCleary W R, Stock A M, et al. Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors[J]. Proc Natl Acad Sci U S A, 1992, 89(2): 718－722.
[22] Bent C J, Isaacs N W, Mitchell T J, et al. Crystal structure of the response regulator 02 receiver domain, the essential YycF two-component system of Streptococcus pneumoniae in both complexed and native states[J]. J Bacteriol, 2004, 186(9): 2 872－2 879.
[23] Zhao H, Heroux A, Sequeira R D, et al. Preliminary crystallographic studies of the regulatory domain of response regulator YycF from an essential two-component signal transduction system[J]. Acta Crystallogr F, 2009, 65(7): 719－722.
[24] Pellecchia M, Montgomery D L, Stevens S Y, et al. Structural insights into substrate binding by the molecular chaperone DnaK[J]. Nat Struct Biol, 2000, 7(4): 298－303.
[25] Stevens S Y, Sanker S, Kent C, et al. Delineation of the allosteric mechanism of a cytidylyltransferase exhibiting negative cooperativity[J]. Nat Struct Biol, 2001, 8(11): 947－952.
[26] Zuiderweg E R. Mapping protein-protein interactions in solution by NMR spectroscopy[J]. Biochemistry, 2002, 41(1): 1－7.
[27] Chang D K, Chien W J, Arunkumar A I. Conformation of a protein kinase C substrate NG(28-43), and its analog in aqueous and sodium dodecyl sulfate micelle solutions[J]. Biophys J, 1997, 72(2 Pt 1): 554-566.
[28] Chen A, Shapiro M J. Affinity NMR[J]. Anal Chem, 1999, 71(19): 669A－675A.
[29] Shuker S B, Hajduk P J, Meadows R P, et al. Discovering high-affinity ligands for proteins: SAR by NMR[J]. Science, 1996, 274(5 292): 1 531－1 534.
[30] van Nuland N A, Kroon G J, Dijkstra K, et al. The NMR determination of the IIA(mtl) binding site on HPr of the Escherichia coli phosphoenol pyruvate-dependent phosphotransferase system[J]. FEBS Lett, 1993, 315(1): 11－15.
[31] Delaglio F, Grzesiek S, Vuister G W, et al. NMRPipe: A multidimensional spectral processing system based on UNIX pipes[J]. J Biomol NMR, 1995, 6(3): 277－293.
[32] Farmer B T, 2nd, Constantine K L, Goldfarb V, et al. Localizing the NADP+ binding site on the MurB enzyme by NMR[J]. Nat Structl Biol, 1996, 3(12): 995－997.
[33] Stock A M, Martinez-Hackert E, Rasmussen B F, et al. Structure of the Mg(2+)-bound form of CheY and mechanism of phosphoryl transfer in bacterial chemotaxis[J]. Biochemistry, 1993, 32(49): 13 375－13 380.
[34] Clore G M, Gronenborn A M, Birdsall B, et al. 19F-n.m.r. studies of 3',5'-difluoromethotrexate binding to Lactobacillus casei dihydrofolate reductase. Molecular motion and coenzyme-induced conformational changes[J]. Biochem J, 1984, 217(3): 659－666.
[35] Lennon A J, Scott N R, Chapman B E, et al. Hemoglobin affinity for 2,3-bisphosphoglycerate in solutions and intact erythrocytes: Studies using pulsed-field gradient nuclear magnetic resonance and Monte Carlo simulations[J]. Biophys J, 1994, 67(5): 2 096－2 109.
[36] Birck C, Chen Y, Hulett F M, et al. The crystal structure of the phosphorylation domain in PhoP reveals a functional tandem association mediated by an asymmetric interface[J]. J Bacteriol, 2003, 185(1): 254－261.
[37] Davies K M, Lowe E D, Venien-Bryan C, et al. The HupR receiver domain crystal structure in its nonphospho and inhibitory phospho states[J]. J Mol Biol, 2009, 385(1): 51－64.
[38] Appleby J L, Bourret R B. Proposed signal transduction role for conserved CheY residue Thr87, a member of the response regulator active-site quintet[J]. J Bacteriol, 1998, 180(14): 3 563－3 569.
[39] Lukat G S, Lee B H, Mottonen J M, et al. Roles of the highly conserved aspartate and lysine residues in the response regulator of bacterial chemotaxis[J]. J Biol Chem, 1991, 266(13): 8 348－8 354.
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