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(epsilon2) atoms and one via its N(delta1). 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 pKa 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.
Hannan
J P
JP
School of Biological Sciences, University of East Anglia, Norwich, UK.
Whittaker
S B
SB
Hemmings
A M
AM
James
R
R
Kleanthous
C
C
Moore
G R
GR
D001665:Binding Sites; D003087:Colicins / Q000737:chemistry* / Q000378:metabolism; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009532:Nickel / Q000378:metabolism*; D017433:Protein Structure, Secondary; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers
D003087:Colicins; D009532:Nickel; D003851:Deoxyribonucleases; D015032:Zinc