Can other metals replace zinc in an estrogen receptor’s DNA-binding domain?

Details

Exposure of bovine estrogen receptor to the metal chelators EDTA and 1,10-phenanthroline caused a loss of nonspecific DNA binding, presumably because they removed zinc from its zinc fingers. In a DNA-cellulose binding assay, nonspecific binding was restored by dialyzing the aporeceptor against buffer containing zinc, cadmium, or cobalt, but not copper or nickel.

For a more detailed study, researchers used a bacterially expressed polypeptide encompassing the DNA-binding domain of the human estrogen receptor. In a mobility shift assay using an oligonucleotide containing a consensus estrogen response element hexamer, the apopolypeptide failed to bind DNA specifically; binding was restored by dialysis against buffer containing zinc, cadmium, or cobalt, but not copper or nickel. The dissociation constants for the estrogen response element hexamer were 66 nM for the zinc-reconstituted polypeptide and 48 nM for the cadmium-reconstituted polypeptide, virtually indistinguishable from the native polypeptide’s Kd of 48 nM; the cobalt-reconstituted polypeptide had lower affinity, with a Kd of 720 nM. Nevertheless, native and zinc-, cadmium-, and cobalt-reconstituted polypeptides gave identical results in a methylation interference assay.

Competition experiments involving zinc and copper or nickel suggested that copper and nickel can bind to zinc finger residues, but do so nonproductively. A zinc blot competition assay established the polypeptide’s relative metal affinities as copper > cadmium > zinc > cobalt > nickel. The researchers concluded that cadmium and cobalt substitution demonstrates structural flexibility in the DNA-binding domain, since the metals have slightly different ionic radii. Subtle differences in DNA-binding affinity or specificity could nevertheless exist and might not be detectable in these experiments. The researchers also suggested that metal substitution in these zinc fingers in vivo might be relevant to the toxicity or carcinogenicity of some of the metals.