pH dependent Ni(II) binding and aggregation of Escherichia coli and Helicobacter pylori NikR.
Résumé
NikR proteins are bacterial metallo-regulatory transcription factors that control the expression of the nickel uptake system and/or nickel containing enzymes such as urease, and are involved in the acid stress response. Here, a comparative study is reported on NikR from Helicobacter pylori (HpNikR) and Escherichia coli (EcNikR), as well as the Q2E mutant of EcNikR. Most attention was focused on the Ni(II) binding properties of these proteins, as a function of pH. The influence of the pH on the Ni(II) binding and aggregation properties was studied using gel filtration analysis and UV-visible absorption spectroscopy in the presence of an increasing concentration of nickel. Q2E and wt EcNikR are identical in Ni(II) binding but the Q2E mutant is impaired to some extent in DNA-binding. For EcNikR it is shown that between pH 6 and 8, addition of Ni(II) above 1 equiv. induces mass aggregation and precipitation, concomitant with binding of Ni(II) up to a maximum of 5-8 Ni(II) ions per monomer. The Ni(II) site with highest affinity is the well-described square planar site with three histidines and one cysteine ligands. Aggregation is complete in the presence of less than 1 extra equiv. of Ni(II) and aggregation is fully reversible and precipitates are rapidly solubilized by addition of EDTA. The sensitivity of EcNikR to aggregation decreases with decreasing pH, concurrent with histidines being the main ligands of the site responsible for aggregation. HpNikR does not display aggregation except at alkaline pH, where 3 Ni(II) equiv. are needed. The participation of a cluster consisting of surface-exposed histidines present in EcNikR but not in HpNikR, is proposed to be involved in aggregation. Our results on HpNikR are compatible with the crystallographic data and with the ability of this protein to bind more than one nickel.