title: Surface plasmon resonance and isothermal titration calorimetry to monitor the Ni(II)-dependent binding of Helicobacter pylori NikR to DNA.
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-OPureAinteraction. An active surface was prepared by affinity capture of OPureAand 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-OPureAcomplex, 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 OPureAurease promoter immobilized on the sensor chip surface.
authors: Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy.
authors: Edoardo
authors: E
authors: Fabini
authors: Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Viale G. Fanin 40, 40127, Bologna, Italy.
authors: Barbara
authors: B
authors: Zambelli
authors: Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Viale G. Fanin 40, 40127, Bologna, Italy.
authors: Luca
authors: L
authors: Mazzei
authors: Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology, University of Bologna, Viale G. Fanin 40, 40127, Bologna, Italy. stefano.ciurli@unibo.it.
authors: Stefano
authors: S
authors: Ciurli
authors: Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy. carlo.bertucci@unibo.it.
authors: Carlo
authors: C
authors: Bertucci
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D055808:Drug Discovery; D016480:Helicobacter pylori / Q000378:metabolism*; D008954:Models, Biological; D009532:Nickel / Q000378:metabolism*; D009875:Operator Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D017186:Titrimetry; D014510:Urease / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; C523724:NikR protein, Helicobacter pylori; D012097:Repressor Proteins; D009532:Nickel; D014510:Urease
keywords: Binding affinity; Helicobacter pylori NikR; ITC; Molecular recognition process; Protein-DNA interaction; SPR