Reactivity of zinc finger cysteines: Chemical modifications within labile zinc fingers in estrogen receptor

Estrogen receptor (ER, alpha isoform) is a 67 kDa zinc finger transcription factor that plays a fundamental role in both normal reproductive gland development and breast carcinogenesis, and also represents a critical molecular target for breast cancer therapy. We are investigating the structural consequences of chemical exposures thought to modify essential zinc finger cysteine residues in human ER. The current study employs mass spectrometry to probe ER zinc finger structural changes induced by a redox-reactive vitamin K3 analog, menadione; a commonly used cysteine alkylator, iodoacetic acid; and a thiol alkylating fluorophore, monobromobimane. Although they are slower to react, the sterically bulkier reagents, monobromobimane and menadione, effectively alkylate the most susceptible ER zinc finger cysteine sulfhydryl groups. Menadione arylation results first in Michael addition of the hydroquinone followed by rapid oxidation to the corresponding quinone, evidenced by a 2 Da mass loss per cysteine residue. Mass spectrometric analysis performed under MALDI conditions reveals both hydroquinone and quinone forms of arylated menadione, whereas only the quinone product is detectable under ESI conditions. Tandem mass spectrometry of a synthetic peptide encompassing the C-terminal half of the structurally more labile second zinc finger of ER (ZnF2B) demonstrates that the two nucleophilic thiols in ZnF2B (Cys-237, Cys-240) are not chemically equivalent in their reactivity to bromobimane or menadione, consistent with their unequal positioning near basic amino acids that affect thiol pKa, thereby rendering Cys-240 more reactive than Cys-237. These findings demonstrate important differential susceptibility of ER zinc finger cysteine residues to thiol reactions.