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

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 Co²⁺, Ni²⁺ and Zn²⁺ to this protein. Data for the Co²⁺-substituted and Ni²⁺-substituted proteins show that the metal ion is coordinated by three histidine residues; and the NMR characteristics of the Ni²⁺-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 Ni²⁺-substituted protein is perturbed by the presence of phosphate, consistent with an X-ray structure showing that phosphate is coordinated to bound Ni²⁺, and by a change in pH, consistent with an ionisable group at the metal centre with a pK(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 Zn²⁺-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 Zn²⁺ does not significantly perturb the spectra, and hence structures, of these conformers beyond the HNH motif region. (C) 2000 Elsevier Science Inc.