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 H NH motif that binds divalent transition metal ions. We have used 1D H-1 and 2D H-1-N-15 NMR methods to characterise the binding of Co2+, Ni2+ and Zn2 to this protein. Data for the Co2+-substituted and Ni2+-substituted protei ns show that the metal ion is coordinated by three histidine residues; and the NMR characteristics of the Ni2+-substituted protein show that two of th e histidines are coordinated through their N-epsilon 2 atoms and one via it s N-delta 1. Furthermore, the NMR spectrum of the Ni2+-substttuted protein is perturbed by the presence of phosphate, consistent with an X-ray structu re 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 reso nances of the metal site, suggesting there is no substantial conformation c hange of the DNase HNH motif on inhibitor binding. H-1-N-15 NMR data for th e Zn2+-substituted DNase show that this protein, like the metal-free DNase, exists as two conformers with different H-1-N-15 correlation NMR spectra, and that the binding of Zn2+ does not significantly perturb the spectra, an d hence structures, of these conformers beyond the HNH motif region. (C) 20 00 Elsevier Science Inc. All rights reserved.