NikR is a prokaryotic Ni2+-dependent transcription factor that regulates the expression of Ni2+-enzymes, such as urease and hydrogenase, and related proteins involved in Ni2+-trafficking. Previous studies have indicated that the homo-tetrameric NikR binds Ni2+ in sites with different affinities, and that this event affects the protein capability to carry out its physiological role by interacting with DNA. However, the modulation of the NikR-DNA binding affinity exerted by different concentrations of Ni2+ and/or additional ions is still unclear. Herein an attempt to clarify this point is described using a multifaceted study of i) the thermodynamic parameters of the interaction between Ni2+ and Helicobacter pylori NikR (HpNikR), ii) the affinity of HpNikR for different metal ions, and iii) the metal ion dependent capability of HpNikR to bind PureA, the promoter of the urease operon. Isothermal titration micro-calorimetry demonstrated that HpNikR binds two Ni2+ ions with a Kd of ca. 10 nM and two additional Ni2+ ions with a Kd of ca. 100 nM in four high-affinity (HA) sites. This metal-protein interaction induces a conformational rearrangement, visible as a slow endothermic event in the ITC raw data, with a kinetic constant in the order of 10-3 s-1. Subsequently, HpNikR binds excess Ni2+ with lower affinity (LA sites), with dissociation constants in the order of μM. ITC demonstrated the absence of specific Ca2+ and Mg2+ binding to the protein, while it established the thermodynamics of the interaction of HpNikR to Zn2+. Mobility shift assays, DNase I footprinting and micro-calorimetry were used to prove that binding of stoichiometric Ni2+ (but not Zn2+) to HA sites (but not to LA sites) selectively activates HpNikR to bind its target urease operator. The thermodynamic parameters of the Ni2+-loaded HpNikR binding to its operator DNA were determined using ITC. These results shed light on which form of the NikR protein is active in regulating the expression of genes belonging to the urease operator in Helicobacter pylori.
Metal and DNA binding properties of NikR from Helicobacter pylori / B. Zambelli; A. Danielli; M. Bellucci; P. Neyroz; S. Romagnoli; V. Scarlato; S. Ciurli. - STAMPA. - (2008), pp. 33-33. (Intervento presentato al convegno Applications of BioCalorimetry tenutosi a Heidelberg (Germania) nel 7-10 Luglio 2008).
Metal and DNA binding properties of NikR from Helicobacter pylori
ZAMBELLI, BARBARA;DANIELLI, ALBERTO;BELLUCCI, MATTEO;NEYROZ, PAOLO;ROMAGNOLI, SIMONA;SCARLATO, VINCENZO;CIURLI, STEFANO LUCIANO
2008
Abstract
NikR is a prokaryotic Ni2+-dependent transcription factor that regulates the expression of Ni2+-enzymes, such as urease and hydrogenase, and related proteins involved in Ni2+-trafficking. Previous studies have indicated that the homo-tetrameric NikR binds Ni2+ in sites with different affinities, and that this event affects the protein capability to carry out its physiological role by interacting with DNA. However, the modulation of the NikR-DNA binding affinity exerted by different concentrations of Ni2+ and/or additional ions is still unclear. Herein an attempt to clarify this point is described using a multifaceted study of i) the thermodynamic parameters of the interaction between Ni2+ and Helicobacter pylori NikR (HpNikR), ii) the affinity of HpNikR for different metal ions, and iii) the metal ion dependent capability of HpNikR to bind PureA, the promoter of the urease operon. Isothermal titration micro-calorimetry demonstrated that HpNikR binds two Ni2+ ions with a Kd of ca. 10 nM and two additional Ni2+ ions with a Kd of ca. 100 nM in four high-affinity (HA) sites. This metal-protein interaction induces a conformational rearrangement, visible as a slow endothermic event in the ITC raw data, with a kinetic constant in the order of 10-3 s-1. Subsequently, HpNikR binds excess Ni2+ with lower affinity (LA sites), with dissociation constants in the order of μM. ITC demonstrated the absence of specific Ca2+ and Mg2+ binding to the protein, while it established the thermodynamics of the interaction of HpNikR to Zn2+. Mobility shift assays, DNase I footprinting and micro-calorimetry were used to prove that binding of stoichiometric Ni2+ (but not Zn2+) to HA sites (but not to LA sites) selectively activates HpNikR to bind its target urease operator. The thermodynamic parameters of the Ni2+-loaded HpNikR binding to its operator DNA were determined using ITC. These results shed light on which form of the NikR protein is active in regulating the expression of genes belonging to the urease operator in Helicobacter pylori.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
