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Bacterial Chemosensing pp 321–335Cite as

Fluorescence Measurement of Kinetics of CheY Autophosphorylation with Small Molecule Phosphodonors

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1729))

Abstract

The Escherichia coli chemotaxis protein CheY is a model receiver domain containing a native tryptophan residue that serves as a fluorescent probe for CheY autophosphorylation with small molecule phosphodonors. Here we describe fluorescence measurement of apparent bimolecular rate constants for reaction of wild type and mutant CheY with phosphodonors acetyl phosphate, phosphoramidate, or monophosphoimidazole. Step-by-step protocols to synthesize phosphoramidate (K+ salt) and monophosphoimidazole (Na+ salt), which are not commercially available, are provided. Key factors to consider in developing autophosphorylation assays for other response regulators are also discussed.

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References

  1. Bourret RB, Silversmith RE (2010) Two-component signal transduction. Curr Opin Microbiol 13:113–115

    Article  Google Scholar 

  2. Zschiedrich CP, Keidel V, Szurmant H (2016) Molecular mechanisms of two-component signal transduction. J Mol Biol 428:3752–3775

    Article  CAS  Google Scholar 

  3. Lukat GS, Stock AM, Stock JB (1990) Divalent metal ion binding to the CheY protein and its significance to phosphotransfer in bacterial chemotaxis. Biochemistry 29:5436–5442

    Article  CAS  Google Scholar 

  4. Needham JV, Chen TY, Falke JJ (1993) Novel ion specificity of a carboxylate cluster Mg(II) binding site: strong charge selectivity and weak size selectivity. Biochemistry 32:3363–3367

    Article  CAS  Google Scholar 

  5. Stock AM, Martinez-Hackert E, Rasmussen BF et al (1993) Structure of the Mg(2+)-bound form of CheY and mechanism of phosphoryl transfer in bacterial chemotaxis. Biochemistry 32:13375–13380

    Article  CAS  Google Scholar 

  6. Appleby JL, Bourret RB (1998) Proposed signal transduction role for conserved CheY residue Thr87, a member of the response regulator active site quintet. J Bacteriol 180:3563–3569

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Lukat GS, Lee BH, Mottonen JM et al (1991) Roles of the highly conserved aspartate and lysine residues in the response regulator of bacterial chemotaxis. J Biol Chem 266:8348–8354

    CAS  PubMed  Google Scholar 

  8. Bourret RB, Hess JF, Simon MI (1990) Conserved aspartate residues and phosphorylation in signal transduction by the chemotaxis protein CheY. Proc Natl Acad Sci U S A 87:41–45

    Article  CAS  Google Scholar 

  9. Hess JF, Bourret RB, Oosawa K et al (1988) Protein phosphorylation and bacterial chemotaxis. Cold Spring Harb Symp Quant Biol 53:41–48

    Article  CAS  Google Scholar 

  10. Cho HS, Lee SY, Yan D et al (2000) NMR structure of activated CheY. J Mol Biol 297:543–551

    Article  CAS  Google Scholar 

  11. Formaneck MS, Ma L, Cui Q (2006) Reconciling the “old” and “new” views of protein allostery: a molecular simulation study of the chemotaxis Y protein (CheY). Proteins 63:846–867

    Article  CAS  Google Scholar 

  12. Lee SY, Cho HS, Pelton JG et al (2001) Crystal structure of activated CheY. Comparison with other activated receiver domains. J Biol Chem 276:16425–16431

    Article  CAS  Google Scholar 

  13. McDonald LR, Boyer JA, Lee AL (2012) Segmental motions, not a two-state concerted switch, underly allostery in CheY. Structure 20:1363–1373

    Article  CAS  Google Scholar 

  14. Lukat GS, McCleary WR, Stock AM et al (1992) Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors. Proc Natl Acad Sci U S A 89:718–722

    Article  CAS  Google Scholar 

  15. Goulian M (2010) Two-component signaling circuit structure and properties. Curr Opin Microbiol 13:184–189

    Article  CAS  Google Scholar 

  16. Page SC, Silversmith RE, Collins EJ et al (2015) Imidazole as a small molecule analogue in two-component signal transduction. Biochemistry 54:7248–7260

    Article  CAS  Google Scholar 

  17. Da Re SS, Deville-Bond D, Tolstykh T et al (1999) Kinetics of CheY phosphorylation by small molecule phosphodonors. FEBS Lett 457:323–326

    Article  Google Scholar 

  18. Immormino RM, Silversmith RE, Bourret RB (2016) A variable active site residue influences the kinetics of response regulator phosphorylation and dephosphorylation. Biochemistry 55:5595–5609

    Article  CAS  Google Scholar 

  19. Immormino RM, Starbird CA, Silversmith RE et al (2015) Probing mechanistic similarities between response regulator signaling proteins and haloacid dehalogenase phosphatases. Biochemistry 54:3514–3527

    Article  CAS  Google Scholar 

  20. Mayover TL, Halkides CJ, Steward RC (1999) Kinetic characterization of CheY phosphorylation reactions: comparison of P-CheA and small-molecule phosphonors. Biochemistry 38:2259–2271

    Article  CAS  Google Scholar 

  21. Silversmith RE, Appleby JL, Bourret RB (1997) Catalytic mechanism of phosphorylation and dephosphorylation of CheY: kinetic characterization of imidazole phosphates as phosphodonors and the role of acid catalysis. Biochemistry 36:14965–14974

    Article  CAS  Google Scholar 

  22. Thomas SA, Immormino RM, Bourret RB et al (2013) Nonconserved active site residues modulate CheY autophosphorylation kinetics and phosphodonor preference. Biochemistry 52:2262–2273

    Article  CAS  Google Scholar 

  23. Wolfe AJ (2010) Physiologically relevant small phosphodonors link metabolism to signal transduction. Curr Opin Microbiol 13:204–209

    Article  CAS  Google Scholar 

  24. Liu W, Hulett FM (1997) Bacillus subtilis PhoP binds to the phoB tandem promoter excusively within the phosphate starvation-inducible promoter. J Bacteriol 179:6302–6310

    Article  CAS  Google Scholar 

  25. Zapf JW, Hoch JA, Whiteley JM (1996) A phosphotransferase activity of the Bacillus subtilis sporulation protein Spo0F that employs phosphoramidate substrates. Biochemistry 35:2926–2933

    Article  CAS  Google Scholar 

  26. Sheridan RC, McCullough JF, Wakefield ZT (1972) Phosphoramidic acid and its salts. Inorg Synth 13:23–26

    Google Scholar 

  27. Rathlev T, Rosenberg T (1956) Non-enzymic formation and rupture of phosphorus to nitrogen linkages in phosphoramido derivatives. Arch Biochem Biophys 65:319–339

    Article  CAS  Google Scholar 

  28. McCleary WR (1996) The activation of PhoB by acetyl phosphate. Mol Microbiol 20:1155–1163

    Article  CAS  Google Scholar 

  29. Creager-Allen RL, Silversmith RE, Bourret RB (2013) A link between dimerization and autophosphorylation of the response regulator PhoB. J Biol Chem 288:21755–21769

    Article  CAS  Google Scholar 

  30. Fernandez I, Otero LH, Klinke S et al (2015) Snapshots of conformational changes shed light into the NtrX receiver domain signal transduction mechanism. J Mol Biol 427:3258–3272

    Article  CAS  Google Scholar 

  31. Ferre A, De La Mora J, Ballado T et al (2004) Biochemical study of multiple CheY response regulators of the chemotactic pathway of Rhodobacter sphaeroides. J Bacteriol 186:5172–5177

    Article  CAS  Google Scholar 

  32. Perron-Savard P, De Crescenzo G, Le Moual H (2005) Dimerization and DNA binding of the Salmonella enterica PhoP response regulator are phosphorylation independent. Microbiology 151:3979–3987

    Article  CAS  Google Scholar 

  33. Pittman MS, Goodwin M, Kelly DJ (2001) Chemotaxis in the human gastric pathogen Helicobacter pylori: different roles for CheW and the three CheV paralogues, and evidence for CheV2 phosphorylation. Microbiology 147:2493–2504

    Article  CAS  Google Scholar 

  34. Guillet V, Ohta N, Cabantous S et al (2002) Crystallographic and biochemical studies of DivK reveal novel features of an essential response regulator in Caulobacter crescentus. J Biol Chem 277:42003–42010

    Article  CAS  Google Scholar 

  35. Madhusudan M, Zapf J, Hoch JA et al (1997) A response regulatory protein with the site of phosphorylation blocked by an arginine interaction: crystal structure of Spo0F from Bacillus subtilis. Biochemistry 36:12739–12745

    Article  CAS  Google Scholar 

  36. Stewart RC, VanBruggen R (2004) Phosphorylation and binding interactions of CheY studied by use of Badan-labeled protein. Biochemistry 43:8766–8777

    Article  CAS  Google Scholar 

  37. Barbieri CM, Stock AM (2008) Universally applicable methods for monitoring response regulator aspartate phosphorylation both in vitro and in vivo using Phos-tag-based reagents. Anal Biochem 376:73–82

    Article  CAS  Google Scholar 

  38. Barbieri CM, Wu T, Stock AM (2013) Comprehensive analysis of OmpR phosphorylation, dimerization, and DNA binding supports a canonical model for activation. J Mol Biol 425:1612–1626

    Article  CAS  Google Scholar 

  39. Buckler DR, Stock AM (2000) Synthesis of [(32)P]phosphoramidate for use as a low molecular weight phosphodonor reagent. Anal Biochem 283:222–227

    Article  CAS  Google Scholar 

  40. Jagadeesan S, Mann P, Schink CW et al (2009) A novel “four-component” signal transduction mechanism regulates developmental progression in Myxococcos xanthus. J Biol Chem 284:21435–21445

    Article  CAS  Google Scholar 

  41. Stadtman ER (1957) Preparation and assay of acetyl phosphate. Methods Enzymol 3:228–231

    Article  Google Scholar 

  42. Feher VA, Zapf JW, Hoch JA et al (1995) 1H, 15N, and 13C backbone chemical shift assignments, secondary structure, and magnesium-binding characteristics of the Bacillus subtilis response regulator, Spo0F, determined by heteronuclear high-resolution NMR. Protein Sci 4:1801–1814

    Article  CAS  Google Scholar 

  43. Sheftic SR, Garcia PP, White E et al (2012) Nuclear magnetic resonance structure and dynamics of the response regulator Sma0114 from Sinorhizobium meliloti. Biochemistry 51:6932–6941

    Article  CAS  Google Scholar 

  44. Zundel CJ, Capener DC, McCleary WR (1998) Analysis of the conserved acidic residues in the regulatory domain of PhoB. FEBS Lett 441:242–246

    Article  CAS  Google Scholar 

  45. Ames SK, Frankema N, Kenney LJ (1999) C-terminal DNA binding stimulates N-terminal phosphorylation of the outer membrane protein regulator OmpR from Escherichia coli. Proc Natl Acad Sci U S A 96:11792–11797

    Article  CAS  Google Scholar 

  46. Schuster M, Silversmith RE, Bourret RB (2001) Conformational coupling in the chemotaxis response regulator CheY. Proc Natl Acad Sci U S A 98:6003–6008

    Article  CAS  Google Scholar 

  47. Barbieri CM, Mack TR, Robinson VL et al (2010) Regulation of response regulator autophosphorylation through interdomain contacts. J Biol Chem 285:32325–32335

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM050860. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Authors and Affiliations

  1. Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC, USA

    Ruth E. Silversmith & Robert B. Bourret

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  1. Ruth E. Silversmith
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  2. Robert B. Bourret
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Correspondence to Robert B. Bourret .

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Editors and Affiliations

  1. Department of Biology, Texas A&M University, College Station, Texas, USA

    Michael D. Manson

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Silversmith, R.E., Bourret, R.B. (2018). Fluorescence Measurement of Kinetics of CheY Autophosphorylation with Small Molecule Phosphodonors. In: Manson, M. (eds) Bacterial Chemosensing. Methods in Molecular Biology, vol 1729. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7577-8_25

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  • DOI: https://doi.org/10.1007/978-1-4939-7577-8_25

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7576-1

  • Online ISBN: 978-1-4939-7577-8

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