Estrogen receptor interaction with immobilized metals: Differential molecular recognition of Zn2+, Cu2+ and Ni2+ and separation of receptor isoforms

Publisher Website
Google Scholar
Abstract
We have utilized iminodiacetate (IDA) gels with immobilized Zn2+, Cu2+ and Ni2+ ions to evaluate the metal binding properties of uterine estrogen receptor proteins. Soluble (cytosol) receptors labeled with [3H]estradiol were analyzed by immobilized metal affinity chromatography (IMAC) before as well as after (1) 3 M urea-induced transformation to the DNA-binding form, and (2) limited trypsin digestion to separate the steroid- and DNA-binding domains. Imidazole (2–200 mM) affinity elution and pH-dependent (pH 7–3.6) elution techniques were both evaluated and found to resolve several receptor isoforms differentially in both the presence and absence of 3 M urea. Individual receptor forms exhibited various affinities for immobilized Zn2+, Cu2+ and Ni2+ ions, but all intact receptor forms were strongly adsorbed to each of the immobilized metals (Ni2+ > Cu2+ » Zn2+) at neutral pH. Generally, similar results were obtained with IDA–Cu2+ and IDA–Ni2+ in the absence of urea. Receptors were tightly bound and not eluted before 100 mM imidazole or pH 3.6. Different results were obtained using IDA–Zn2+; at least four receptor isoforms were resolved on IDA–Zn2+. Receptor–metal interaction heterogeneity and affinity for IDA–Zn2+ and IDA–Cu2+, but not IDA–Ni2+, were substantially decreased in the presence of 3 M urea. The receptor isoforms identified and separated by IDA–Zn2+ chromatography were not separable using high-performance size-exclusion chromatography, density gradient centrifugation, chromatofocusing or DNA-affinity chromatography. The affinity of trypsin-generated (mero) receptor forms for each of the immobilized metals was decreased relative to that of intact receptor. High-affinity metal-binding sites were mapped to the DNA-binding domain, but at least one of the metal-binding sites is located on the steroidbinding domain. Recovery of all receptor forms from the immobilized metal ion columns was routinely above 90%. These results demonstrate the differential utility of various immobilized metals to characterize and separate individual receptor isoforms and domain structures. Receptor-metal interactions warrant further investigation to establish their effects on receptor structure/function relationships. In addition to the biological implications, recognition of estrogen receptor proteins as metal-binding proteins suggests new and potentially powerful receptor immobilization and purification regimes previously unexplored by those in this field.