title: Spectroscopic determination of the binding affinity of zinc to the DNA-binding domains of nuclear hormone receptors.
abstract: 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.
authors.affiliation: Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Payne
authors.forename: Brian W
authors.initials: BW
authors.lastname: Rous
authors.forename: Adam L
authors.initials: AL
authors.lastname: Tenderholt
authors.forename: Hilary Arnold
authors.initials: HA
authors.lastname: Godwin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D016384:Consensus Sequence / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D047628:Estrogen Receptor alpha; D005822:Genetic Vectors; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D011960:Receptors, Estrogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D008024:Ligands; D010446:Peptide Fragments; D011960:Receptors, Estrogen; D011965:Receptors, Glucocorticoid; D003035:Cobalt; D015032:Zinc