NMR studies of metal ion binding to the Zn-finger-like HNH motif of colicin E9

Abstract

The 134 amino acid DNase domain of colicin E9 contains a zinc-finger-like HNH motif that binds divalent transition metal ions. We have used 1D 1H and 2D 1H– 15N NMR methods to characterise the binding of Co 2+, Ni 2+ and Zn 2+ to this protein. Data for the Co 2+-substituted and Ni 2+-substituted proteins show that the metal ion is coordinated by three histidine residues; and the NMR characteristics of the Ni 2+-substituted protein show that two of the histidines are coordinated through their N ε2 atoms and one via its N δ1. Furthermore, the NMR spectrum of the Ni 2+-substituted protein is perturbed by the presence of phosphate, consistent with an X-ray structure showing that phosphate is coordinated to bound Ni 2+, and by a change in pH, consistent with an ionisable group at the metal centre with a p K a of 7.9. Binding of an inhibitor protein to the DNase does not perturb the resonances of the metal site, suggesting there is no substantial conformation change of the DNase HNH motif on inhibitor binding. 1H– 15N NMR data for the Zn 2+-substituted DNase show that this protein, like the metal-free DNase, exists as two conformers with different 1H– 15N correlation NMR spectra, and that the binding of Zn 2+ does not significantly perturb the spectra, and hence structures, of these conformers beyond the HNH motif region.