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 Co2+ 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 Co2+ to the two proteins was determined relative to the binding affinity of Co2+ to the zinc finger consensus peptide, CP-1. Using the known dissociation constant of Co2+ 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(-1) M. Similarly, the dissociation constants of Co2+ from GR-DBD were calculated: K-d1Co = 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 Zn2+ displaces Co2+ from the metal-binding sites of hERalpha-DBD and GR-DBD indicate that Zn2+ binds to each of the Cys(4) metal-binding sites approximately 3 orders of magnitude more tightly than Co2+ 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., Hemdridez, 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.