Surface plasmon resonance and isothermal titration calorimetry to monitor the Ni(II)-dependent binding of Helicobacter pylori NikR to DNA
- 538 Downloads
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.
KeywordsMolecular 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.
- 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
- 8.Maroney MJ, Ciurli S. Nonredox nickel enzymes. Chem Rev. 2013.Google Scholar
- 17.Yan C, Higgins PJ. Drugging the undruggable: transcription therapy for cancer. Biochim Biophys Acta. 2013;1835(1):76–85.Google Scholar
- 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
- 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.Hansen LD, Transtrum MK, Quinn C, Demarse N. Enzyme-catalyzed and binding reaction kinetics determined by titration calorimetry. Biochim Biophys Acta. 2015.Google Scholar
- 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
- 39.de Mol NJ, Fischer MJE. Surface plasmon resonance; methods and protocols. Sci-Tech News, vol 4. Springer; 2010, p. 53–66.Google Scholar