Spectroscopic determination of the binding affinity of zinc to the DNA-binding domains of nuclear hormone receptors

Zinc binding to the two Cys(4) sites present in the DNA-binding domain (DBD) of nuclear hormone receptor proteins is required for proper folding of the domain and for protein activity. By utilizing Co(2+) as a spectroscopic probe, we have characterized the metal-binding properties of the two Cys(4) structural zinc-binding sites found in the DBD of human estrogen receptor alpha (hERalpha-DBD) and rat glucocorticoid receptor (GR-DBD). The binding affinity of Co(2+) to the two proteins was determined relative to the binding affinity of Co(2+) to the zinc finger consensus peptide, CP-1. Using the known dissociation constant of Co(2+) from CP-1, the dissociation constants of cobalt from hERalpha-DBD were calculated: K(d1)(Co) = 2.2 (+/- 1.0) x 10(-7) M and K(d2)(Co) = 6.1 (+/- 1.5) x 10(-7) M. Similarly, the dissociation constants of Co(2+) from GR-DBD were calculated: K(d1)(Co) = 4.1 (+/- 0.6) x 10(-7) M and K(d2)(Co) = 1.7 (+/- 0.3) x 10(-7) M. Metal-binding studies conducted in which Zn(2+) displaces Co(2+) from the metal-binding sites of hERalpha-DBD and GR-DBD indicate that Zn(2+) binds to each of the Cys(4) metal-binding sites approximately 3 orders of magnitude more tightly than Co(2+) does: the stoichiometric dissociation constants are K(d1)(Zn) = 1 (+/- 1) x 10(-10) M and K(d2)(Zn) = 5 (+/- 1) x 10(-10) M for hERalpha-DBD and K(d1)(Zn) = 2 (+/- 1) x 10(-10) M and K(d2)(Zn) = 3 (+/- 1) x 10(-10) M for GR-DBD. These affinities are comparable to those observed for most other naturally occurring structural zinc-binding sites. In contrast to the recent prediction by Low et. al. that zinc binding in these systems should be cooperative Low, L. Y., Hernández, H., Robinson, C. V., O'Brien, R., Grossmann, J. G., Ladbury, J. E., and Luisi, B. (2002) J. Mol. Biol. 319, 87-106], these data suggest that the zincs that bind to the two sites in the DBDs of hERalpha-DBD and GR-DBD do not interact.