Preferential oxidation of zinc finger 2 in estrogen receptor DNA-binding domain prevents dimerization and, hence, DNA binding

For approximately one-third of estrogen receptor (ER)-positive breast cance r patients, extracted tumor ER is unable to bind to its cognate DNA estroge n response element (ERE), an effect that is partly reversible by the thiol- reducing agent dithiothreitol (DTT). Full-length (67 kDa) ER or its 11 kDa recombinant DNA-binding domain (ER-DBD) is also susceptible to loss of stru cture and function by the action of oxidants such as diamide and hydrogen p eroxide; however, prior DNA binding by ER or ER-DBD protects against this o xidant induced loss of function. The ER-DBD contains two (Cys)(4)-liganded zinc finger motifs that cooperate to stabilize a rigid DNA-binding recognit ion helix and a flexible helix-supported dimerization loop, respectively. C omparisons between synthetic peptide analogues of each zinc finger and reco mbinant ER-DBD in the presence of zinc by electrophoretic mobility shift as say, circular dichroism, and mass spectrometry confirm that cooperativity b etween these zinc fingers is required for both ER-DBD structure (alpha-heli city) and function (dimeric DNA binding). Rapid proteolytic digestion of mo nomeric, non-DNA-bound ER-DBD followed by HPLC-MS analysis of the resulting peptides demonstrates that zinc inhibits thiol oxidation of the DNA-bindin g finger, but not the finger supporting the flexible dimerization loop, whi ch remains sensitive to internal disulfide formation. These findings indica te that the loss of ER DNA-binding function in extracts from some primary b reast tumors and in ER or ER-DBD exposed to thiol-reacting oxidants results from this asymmetric zinc finger susceptibility to disulfide formation tha t prevents dimerization. Although ER-DBD contains several strategically loc ated methionine residues, they are less susceptible to oxidation than the t hiol groups and, thus, afford no protection against cysteine oxidation and consequent loss of ER DNA-binding function.