Analytical and Bioanalytical Chemistry

, Volume 408, Issue 28, pp 7971–7980 | Cite as

Surface plasmon resonance and isothermal titration calorimetry to monitor the Ni(II)-dependent binding of Helicobacter pylori NikR to DNA

  • Edoardo Fabini
  • Barbara Zambelli
  • Luca Mazzei
  • Stefano CiurliEmail author
  • Carlo BertucciEmail author
Research Paper


NikR is a transcription factor that regulates the expression of Ni(II)-dependent enzymes and other proteins involved in nickel trafficking. In the human pathogenic bacterium Helicobacter pylori, NikR (HpNikR) controls, among others, the expression of the Ni(II) enzyme urease by binding the double-strand DNA (dsDNA) operator region of the urease promoter (OP ureA ) in a Ni(II)-dependent mode. This article describes the complementary use of surface plasmon resonance (SPR) spectroscopy and isothermal titration calorimetry (ITC) to carry out a mechanistic characterization of the HpNikR–OP ureA interaction. An active surface was prepared by affinity capture of OP ureA and validated for the recognition process in the SPR experiments. Subsequently, the Ni(II)-dependent affinity of the transcription factor for its operator region was assessed through kinetic evaluation of the binding process at variable Ni(II) concentrations. The kinetic data are consistent with a two-step binding mode involving an initial encounter between the two interactants, followed by a conformational rearrangement of the HpNikR–OP ureA complex, leading to high affinity binding. This conformational change is only observed in the presence of the full set of four Ni(II) ions bound to the protein. The SPR assay developed and validated in this study constitutes a suitable method to screen potential drug lead candidates acting as inhibitors of this protein–dsDNA interaction.

Graphical Abstract

Pictorial representation of the interaction between HpNikR, flowing in solution, and the OP ureA urease promoter immobilized on the sensor chip surface


Molecular recognition process Protein-DNA interaction Binding affinity Helicobacter pylori NikR SPR ITC 


Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2016_9894_MOESM1_ESM.pdf (684 kb)
ESM 1 (PDF 683 kb)


  1. 1.
    Zambelli B, Ciurli S. Nickel and human health. Met Ions Life Sci. 2013;13:321–57.CrossRefGoogle Scholar
  2. 2.
    Chivers PT. Cobalt and nickel. In: Maret W, Wedd A, editors. Binding, transport and storage of metal ions in biological cells. The Royal Society of Chemistry; 2014. p. 381–428.Google Scholar
  3. 3.
    Musiani F, Zambelli B, Bazzani M, Mazzei L, Ciurli S. Nickel-responsive transcriptional regulators. Metallomics. 2015;7(9):1305–18.CrossRefGoogle Scholar
  4. 4.
    van Vliet AH, Poppelaars SW, Davies BJ, Stoof J, Bereswill S, Kist M, et al. NikR mediates nickel-responsive transcriptional induction of urease expression in Helicobacter pylori. Infect Immun. 2002;70(6):2846–52.CrossRefGoogle Scholar
  5. 5.
    Contreras M, Thiberge JM, Mandrand-Berthelot MA, Labigne A. Characterization of the roles of NikR, a nickel-responsive pleiotropic autoregulator of Helicobacter pylori. Mol Microbiol. 2003;49(4):947–63.CrossRefGoogle Scholar
  6. 6.
    Muller C, Bahlawane C, Aubert S, Delay CM, Schauer K, Michaud-Soret I, et al. Hierarchical regulation of the NikR-mediated nickel response in Helicobacter pylori. Nucleic Acids Res. 2011;39(17):7564–75.CrossRefGoogle Scholar
  7. 7.
    van Vliet AH, Ernst FD, Kusters JG. NikR-mediated regulation of Helicobacter pylori acid adaptation. Trends Microbiol. 2004;12(11):489–94.CrossRefGoogle Scholar
  8. 8.
    Maroney MJ, Ciurli S. Nonredox nickel enzymes. Chem Rev. 2013.Google Scholar
  9. 9.
    Schreiter ER, Sintchak MD, Guo Y, Chivers PT, Sauer RT, Drennan CL. Crystal structure of the nickel-responsive transcription factor NikR. Nat Struct Biol. 2003;10(10):794–9.CrossRefGoogle Scholar
  10. 10.
    Chivers PT, Tahirov TH. Structure of Pyrococcus horikoshii NikR: nickel sensing and implications for the regulation of DNA recognition. J Mol Biol. 2005;348(3):597–607.CrossRefGoogle Scholar
  11. 11.
    Schreiter ER, Wang SC, Zamble DB, Drennan CL. NikR-operator complex structure and the mechanism of repressor activation by metal ions. Proc Natl Acad Sci U S A. 2006;103(37):13676–81.CrossRefGoogle Scholar
  12. 12.
    Dian C, Schauer K, Kapp U, McSweeney SM, Labigne A, Terradot L. Structural basis of the nickel response in Helicobacter pylori: crystal structures of HpNikR in Apo and nickel-bound states. J Mol Biol. 2006;361(4):715–30.CrossRefGoogle Scholar
  13. 13.
    Phillips CM, Schreiter ER, Guo Y, Wang SC, Zamble DB, Drennan CL. Structural basis of the metal specificity for nickel regulatory protein NikR. Biochemistry. 2008;47(7):1938–46.CrossRefGoogle Scholar
  14. 14.
    West AL, St John F, Lopes PE, MacKerell Jr AD, Pozharski E, Michel SL. Holo-Ni(II)HpNikR is an asymmetric tetramer containing two different nickel-binding sites. J Am Chem Soc. 2010;132(41):14447–56.CrossRefGoogle Scholar
  15. 15.
    Benini S, Cianci M, Ciurli S. Holo-Ni2+ Helicobacter pylori NikR contains four square-planar nickel-binding sites at physiological pH. Dalton Trans. 2011;40(31):7831–3.CrossRefGoogle Scholar
  16. 16.
    Kosikowska P, Berlicki L. Urease inhibitors as potential drugs for gastric and urinary tract infections: a patent review. Expert Opin Ther Pat. 2011;21(6):945–57.CrossRefGoogle Scholar
  17. 17.
    Yan C, Higgins PJ. Drugging the undruggable: transcription therapy for cancer. Biochim Biophys Acta. 2013;1835(1):76–85.Google Scholar
  18. 18.
    Lazo JS, Sharlow ER. Drugging undruggable molecular cancer targets. Annu Rev Pharmacol Toxicol. 2016;56:23–40.CrossRefGoogle Scholar
  19. 19.
    Chan LL, Pineda M, Heeres JT, Hergenrother PJ, Cunningham BT. A general method for discovering inhibitors of protein-DNA interactions using photonic crystal biosensors. ACS Chem Biol. 2008;3(7):437–48.CrossRefGoogle Scholar
  20. 20.
    Alonso N, Guillen R, Chambers JW, Leng F. A rapid and sensitive high-throughput screening method to identify compounds targeting protein-nucleic acids interactions. Nucleic Acids Res. 2015;43(8), e52.CrossRefGoogle Scholar
  21. 21.
    Cooper MA. Optical biosensors in drug discovery. Nat Rev Drug Discov. 2002;1(7):515–28.CrossRefGoogle Scholar
  22. 22.
    Rich RL, Myszka DG. Higher-throughput, label-free, real-time molecular interaction analysis. Anal Biochem. 2007;361(1):1–6.CrossRefGoogle Scholar
  23. 23.
    Nguyen HH, Park J, Kang S, Kim M. Surface plasmon resonance: a versatile technique for biosensor applications. Sensors. 2015;15(5):10481–510.CrossRefGoogle Scholar
  24. 24.
    Myszka DG, Abdiche YN, Arisaka F, Byron O, Eisenstein E, Hensley P, et al. The ABRF-MIRG’02 study: assembly state, thermodynamic, and kinetic analysis of an enzyme/inhibitor interaction. J Biomol Tech. 2003;14(4):247–69.Google Scholar
  25. 25.
    Geitmann M, Danielson UH. Studies of substrate-induced conformational changes in human cytomegalovirus protease using optical biosensor technology. Anal Biochem. 2004;332(2):203–14.CrossRefGoogle Scholar
  26. 26.
    Seeger C, Gorny X, Reddy PP, Seidenbecher C, Danielson UH. Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function. J Mol Recognit. 2012;25(10):495–503.CrossRefGoogle Scholar
  27. 27.
    Nordstrom H, Gossas T, Hamalainen M, Kallblad P, Nystrom S, Wallberg H, et al. Identification of MMP-12 inhibitors by using biosensor-based screening of a fragment library. J Med Chem. 2008;51(12):3449–59.CrossRefGoogle Scholar
  28. 28.
    Wear MA, Patterson A, Malone K, Dunsmore C, Turner NJ, Walkinshaw MD. A surface plasmon resonance-based assay for small molecule inhibitors of human cyclophilin A. Anal Biochem. 2005;345(2):214–26.CrossRefGoogle Scholar
  29. 29.
    Peserico A, Germani A, Sanese P, Barbosa AJ, di Virgilio V, Fittipaldi R, et al. A SMYD3 small-molecule inhibitor impairing cancer cell growth. J Cell Physiol. 2015;230(10):2447–60.CrossRefGoogle Scholar
  30. 30.
    Ghai R, Falconer RJ, Collins BM. Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010. J Mol Recognit. 2012;25(1):32–52.CrossRefGoogle Scholar
  31. 31.
    Mazzei L, Ciurli S, Zambelli B. Hot biological catalysis: isothermal titration calorimetry to characterize enzymatic reactions. J Vis Exp. 2014;(86).Google Scholar
  32. 32.
    Hansen LD, Transtrum MK, Quinn C, Demarse N. Enzyme-catalyzed and binding reaction kinetics determined by titration calorimetry. Biochim Biophys Acta. 2015.Google Scholar
  33. 33.
    Mazzei L, Ciurli S, Zambelli B. Isothermal titration calorimetry to characterize enzymatic reactions. Methods Enzymol. 2016;567:215–36.CrossRefGoogle Scholar
  34. 34.
    Burnouf D, Ennifar E, Guedich S, Puffer B, Hoffmann G, Bec G, et al. kinITC: a new method for obtaining joint thermodynamic and kinetic data by isothermal titration calorimetry. J Am Chem Soc. 2012;134(1):559–65.CrossRefGoogle Scholar
  35. 35.
    Dumas P, Ennifar E, Da Veiga C, Bec G, Palau W, Di Primo C, et al. Extending ITC to kinetics with kinITC. Methods Enzymol. 2016;567:157–80.CrossRefGoogle Scholar
  36. 36.
    Guedich S, Puffer-Enders B, Baltzinger M, Hoffmann G, Da Veiga C, Jossinet F et al. Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches. RNA Biol. 2016;1–18.Google Scholar
  37. 37.
    Zambelli B, Danielli A, Romagnoli S, Neyroz P, Ciurli S, Scarlato V. High-affinity Ni2+ binding selectively promotes binding of Helicobacter pylori NikR to its target urease promoter. J Mol Biol. 2008;383(5):1129–43.CrossRefGoogle Scholar
  38. 38.
    Zambelli B, Bellucci M, Danielli A, Scarlato V, Ciurli S. The Ni2+ binding properties of Helicobacter pylori NikR. Chem Commun. 2007;35:3649–51.CrossRefGoogle Scholar
  39. 39.
    de Mol NJ, Fischer MJE. Surface plasmon resonance; methods and protocols. Sci-Tech News, vol 4. Springer; 2010, p. 53–66.Google Scholar
  40. 40.
    Karlsson R, Katsamba PS, Nordin H, Pol E, Myszka DG. Analyzing a kinetic titration series using affinity biosensors. Anal Biochem. 2006;349(1):136–47.CrossRefGoogle Scholar
  41. 41.
    Myszka DG. Improving biosensor analysis. J Mol Recognit. 1999;12(5):279–84.CrossRefGoogle Scholar
  42. 42.
    Scheuermann TH, Brautigam CA. High-precision, automated integration of multiple isothermal titration calorimetric thermograms: new features of NITPIC. Methods. 2015;76:87–98.CrossRefGoogle Scholar
  43. 43.
    Karlsson R, Fält A. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J Immunol Methods. 1997;200(1–2):121–33.CrossRefGoogle Scholar
  44. 44.
    Rich RL, Myszka DG. Grading the commercial optical biosensor literature-class of 2008: ‘The Mighty Binders’. J Mol Recognit. 2010;23(1):1–64.CrossRefGoogle Scholar
  45. 45.
    Bahlawane C, Dian C, Muller C, Round A, Fauquant C, Schauer K, et al. Structural and mechanistic insights into Helicobacter pylori NikR activation. Nucleic Acids Res. 2010;38(9):3106–18.CrossRefGoogle Scholar
  46. 46.
    West AL, Evans SE, Gonzalez JM, Carter LG, Tsuruta H, Pozharski E, et al. Ni(II) coordination to mixed sites modulates DNA binding of HpNikR via a long-range effect. Proc Natl Acad Sci U S A. 2012;109(15):5633–8.CrossRefGoogle Scholar
  47. 47.
    Musiani F, Bertosa B, Magistrato A, Zambelli B, Turano P, Losasso V, et al. Computational study of the DNA-binding protein Helicobacter pylori NikR: the role of Ni(2+). J Chem Theory Comput. 2010;6(11):3503–15.CrossRefGoogle Scholar
  48. 48.
    Mazzei L, Dobrovolska O, Musiani F, Zambelli B, Ciurli S. On the interaction of Helicobacter pylori NikR, a Ni(II)-responsive transcription factor, with the urease operator: in solution and in silico studies. J Biol Inorg Chem. 2015;20(6):1021–37.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Department of Pharmacy and BiotechnologyUniversity of BolognaBolognaItaly
  2. 2.Laboratory of Bioinorganic Chemistry, Department of Pharmacy and BiotechnologyUniversity of BolognaBolognaItaly

Personalised recommendations