Analytical and Bioanalytical Chemistry

, Volume 408, Issue 21, pp 5925–5933 | Cite as

ATP and autophosphorylation driven conformational changes of HipA kinase revealed by ion mobility and crosslinking mass spectrometry

Research Paper

Abstract

Toxin-antitoxin systems are genetic modules involved in a broad range of bacterial cellular processes including persistence, multidrug resistance and tolerance, biofilm formation, and pathogenesis. In type II toxin-antitoxin systems, both the toxin and antitoxin are proteins. In the prototypic Escherichia coli HipA-HipB module, the antitoxin HipB forms a complex with the protein kinase HipA and sequesters it in the nucleoid. HipA is then no longer able to phosphorylate glutamyl-tRNA-synthetase and this prevents the initiation of the forthcoming stringent response. Here we investigated the assembly of the Shewanella oneidensis MR-1 HipA-HipB complex using native electrospray ion mobility-mass spectrometry and chemical crosslinking combined with mass spectrometry. We revealed that the HipA autophosphorylation was accompanied by a large conformational change, and confirmed structural evidence that S. oneidensis MR-1 HipA-HipB assembly was distinct from the prototypic E. coli HipA-HipB complex.

Graphical abstract

Ion mobility mass spectrometry shows a two phase transition from unstructured HipA to a compact folded phosphorylated protein

Keywords

Toxin-antitoxin system HipAB Ion mobility Chemical crosslinking Mass spectrometry 

Supplementary material

216_2016_9709_MOESM1_ESM.pdf (177 kb)
ESM 1(PDF 177 kb)

References

  1. 1.
    Hau HH, Gralnick JA. Ecology and biotechnology of the genus Shewanella. Annu Rev Microbiol. 2007;61:237–58.CrossRefGoogle Scholar
  2. 2.
    El-Naggar MY, Wanger G, Leung KM, Yuzvinsky TD, Southam G, Yang J, et al. Electrical transport along bacterial nanowires from Shewanella oneidensis MR-1. Proc Natl Acad Sci U S A. 2010;107(42):18127–31.CrossRefGoogle Scholar
  3. 3.
    Gorby YA, Yanina S, McLean JS, Rosso KM, Moyles D, Dohnalkova A, et al. Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc Natl Acad Sci U S A. 2006;103(30):11358–63.CrossRefGoogle Scholar
  4. 4.
    Theunissen S, De Smet L, Dansercoer A, Motte B, Coenye T, Van Beeumen JJ, et al. The 285 kDa Bap/RTX hybrid cell surface protein (SO4317) of Shewanella oneidensis MR-1 is a key mediator of biofilm formation. Res Microbiol. 2010;161(2):144–52.CrossRefGoogle Scholar
  5. 5.
    Correia FF, D’Onofrio A, Rejtar T, Li L, Karger BL, Makarova K, et al. Kinase activity of overexpressed HipA is required for growth arrest and multidrug tolerance in Escherichia coli. J Bacteriol. 2006;188(24):8360–7.CrossRefGoogle Scholar
  6. 6.
    Wen Y, Behiels E, Felix J, Elegheert J, Vergauwen B, Devreese B, et al. The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence. Nucleic Acids Res. 2014;42(15):10134–47.CrossRefGoogle Scholar
  7. 7.
    Schumacher MA, Piro KM, Xu W, Hansen S, Lewis K, Brennan RG. Molecular mechanisms of HipA-mediated multidrug tolerance and its neutralization by HipB. Science. 2009;323(5912):396–401.CrossRefGoogle Scholar
  8. 8.
    Schumacher MA, Min J, Link TM, Guan Z, Xu W, Ahn YH, et al. Role of unusual P loop ejection and autophosphorylation in HipA-mediated persistence and multidrug tolerance. Cell Rep. 2012;2(3):518–25.CrossRefGoogle Scholar
  9. 9.
    Germain E, Castro-Roa D, Zenkin N, Gerdes K. Molecular mechanism of bacterial persistence by HipA. Mol Cell. 2013;52(2):248–54.CrossRefGoogle Scholar
  10. 10.
    Kaspy I, Rotem E, Weiss N, Ronin I, Balaban NQ, Glaser G. HipA-mediated antibiotic persistence via phosphorylation of the glutamyl-tRNA-synthetase. Nat Commun. 2013;4:3001.CrossRefGoogle Scholar
  11. 11.
    Li T, Yin N, Liu H, Pei J, Lai L. Novel inhibitors of toxin HipA reduce multidrug tolerant persisters. ACS Med Chem Lett. 2016;7(5):449–53.CrossRefGoogle Scholar
  12. 12.
    Hopper JT, Oldham NJ. Collision induced unfolding of protein ions in the gas phase studied by ion mobility-mass spectrometry: the effect of ligand binding on conformational stability. J Am Soc Mass Spectrom. 2009;20(10):1851–8.CrossRefGoogle Scholar
  13. 13.
    Hyung SJ, Robinson CV, Ruotolo BT. Gas-phase unfolding and disassembly reveals stability differences in ligand-bound multiprotein complexes. Chem Biol. 2009;16(4):382–90.CrossRefGoogle Scholar
  14. 14.
    Schumacher MA, Balani P, Min J, Chinnam NB, Hansen S, Vulic M, et al. HipBA-promoter structures reveal the basis of heritable multidrug tolerance. Nature. 2015;524(7563):59–64.CrossRefGoogle Scholar
  15. 15.
    Black DS, Irwin B, Moyed HS. Autoregulation of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis. J Bacteriol. 1994;176(13):4081–91.Google Scholar
  16. 16.
    Ruotolo BT, Benesch JL, Sandercock AM, Hyung SJ, Robinson CV. Ion mobility-mass spectrometry analysis of large protein complexes. Nat Protoc. 2008;3(7):1139–52.CrossRefGoogle Scholar
  17. 17.
    Benesch JL, Ruotolo BT. Mass spectrometry: come of age for structural and dynamical biology. Curr Opin Struct Biol. 2011;21(5):641–9.CrossRefGoogle Scholar
  18. 18.
    Konijnenberg A, Butterer A, Sobott F. Native ion mobility-mass spectrometry and related methods in structural biology. Biochim Biophys Acta. 2013;1834(6):1239–56.CrossRefGoogle Scholar
  19. 19.
    Leitner A, Joachimiak LA, Bracher A, Monkemeyer L, Walzthoeni T, Chen B, et al. The molecular architecture of the eukaryotic chaperonin TRiC/CCT. Structure. 2012;20(5):814–25.CrossRefGoogle Scholar
  20. 20.
    Walzthoeni T, Claassen M, Leitner A, Herzog F, Bohn S, Forster F, et al. False discovery rate estimation for cross-linked peptides identified by mass spectrometry. Nat Methods. 2012;9(9):901–3.CrossRefGoogle Scholar
  21. 21.
    Yang B, Wu YJ, Zhu M, Fan SB, Lin J, Zhang K, et al. Identification of cross-linked peptides from complex samples. Nat Methods. 2012;9(9):904–6.CrossRefGoogle Scholar
  22. 22.
    De Gieter S, Konijnenberg A, Talavera A, Butterer A, Haesaerts S, De Greve H, et al. The intrinsically disordered domain of the antitoxin Phd chaperones the toxin Doc against irreversible inactivation and misfolding. J Biol Chem. 2014;289(49):34013–23.CrossRefGoogle Scholar
  23. 23.
    Pacholarz KJ, Garlish RA, Taylor RJ, Barran PE. Mass spectrometry based tools to investigate protein-ligand interactions for drug discovery. Chem Soc Rev. 2012;41(11):4335–55.CrossRefGoogle Scholar
  24. 24.
    Niu S, Rabuck JN, Ruotolo BT. Ion mobility-mass spectrometry of intact protein-ligand complexes for pharmaceutical drug discovery and development. Curr Opin Chem Biol. 2013;17(5):809–17.CrossRefGoogle Scholar
  25. 25.
    Williams JJ, Hergenrother PJ. Artificial activation of toxin-antitoxin systems as an antibacterial strategy. Trends Microbiol. 2012;20(6):291–8.CrossRefGoogle Scholar
  26. 26.
    Shapiro S. Speculative strategies for new antibacterials: all roads should not lead to Rome. J Antibiot. 2013;66(7):371–86.CrossRefGoogle Scholar
  27. 27.
    Wen Y, Behiels E, Devreese B. Toxin-antitoxin systems: their role in persistence, biofilm formation, and pathogenicity. Pathogens Dis. 2014;70(3):240–9.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Center for Translational Medicine, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and TechnologyXi’an Jiaotong UniversityXi’anChina
  2. 2.Unit for Biological Mass Spectrometry and Proteomics, Laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE)Ghent UniversityGhentBelgium
  3. 3.Biomolecular & Analytical Mass Spectrometry Group and UA-VITO Center for Proteomics (CFP-CEPROMA)University of AntwerpAntwerpBelgium

Personalised recommendations