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

TY - JOUR

T1 - NMR studies of metal ion binding to the Zn-finger-like HNH motif of colicin E9

AU - Hannan, J P

AU - Whittaker, S B

AU - Hemmings, A M

AU - James, R

AU - Kleanthous, C

AU - Moore, G R

AU - Kleanthous, Colin

PY - 2000

Y1 - 2000

N2 - 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.

AB - 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.

KW - Binding Sites

KW - Colicins

KW - Deoxyribonucleases

KW - Escherichia coli

KW - Hydrogen-Ion Concentration

KW - Magnetic Resonance Spectroscopy

KW - Models, Molecular

KW - Nickel

KW - Protein Structure, Secondary

KW - Zinc

KW - Zinc Fingers

M3 - Article

C2 - 10830890

SN - 0162-0134

VL - 79

SP - 365

EP - 370

JO - JOURNAL OF INORGANIC BIOCHEMISTRY

JF - JOURNAL OF INORGANIC BIOCHEMISTRY

IS - 1-4

ER -