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 (OPureA) 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–OPureA interaction. An active surface was prepared by affinity capture of OPureA 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–OPureA 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. [Figure not available: see fulltext.]

Surface plasmon resonance and isothermal titration calorimetry to monitor the Ni(II)-dependent binding of Helicobacter pylori NikR to DNA / Fabini, Edoardo; Zambelli, Barbara; Mazzei, Luca; Ciurli, STEFANO LUCIANO; Bertucci, Carlo. - In: ANALYTICAL AND BIOANALYTICAL CHEMISTRY. - ISSN 1618-2642. - STAMPA. - 408:28(2016), pp. 7971-7980. [10.1007/s00216-016-9894-0]

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

FABINI, EDOARDO;ZAMBELLI, BARBARA;MAZZEI, LUCA;CIURLI, STEFANO LUCIANO;BERTUCCI, CARLO
2016

Abstract

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 (OPureA) 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–OPureA interaction. An active surface was prepared by affinity capture of OPureA 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–OPureA 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. [Figure not available: see fulltext.]
2016
Surface plasmon resonance and isothermal titration calorimetry to monitor the Ni(II)-dependent binding of Helicobacter pylori NikR to DNA / Fabini, Edoardo; Zambelli, Barbara; Mazzei, Luca; Ciurli, STEFANO LUCIANO; Bertucci, Carlo. - In: ANALYTICAL AND BIOANALYTICAL CHEMISTRY. - ISSN 1618-2642. - STAMPA. - 408:28(2016), pp. 7971-7980. [10.1007/s00216-016-9894-0]
Fabini, Edoardo; Zambelli, Barbara; Mazzei, Luca; Ciurli, STEFANO LUCIANO; Bertucci, Carlo
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/569980
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