Estrogen receptor interaction with immobilized metals: differential molecular recognition of Zn2+, Cu2+ and Ni2+ and separation of receptor isoforms.
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+ greater than Cu2+ much greater than 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 steroid-binding 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.
Hutchens
T W
TW
Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.
Li
C M
CM
D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent; D002417:Cattle; D002846:Chromatography, Affinity; D003300:Copper / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism; D066298:In Vitro Techniques; D009532:Nickel / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011960:Receptors, Estrogen / Q000378:metabolism*; D012995:Solubility; D014357:Trypsin / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology; D015032:Zinc / Q000378:metabolism*
D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D011960:Receptors, Estrogen; D004958:Estradiol; D003300:Copper; D009532:Nickel; D014508:Urea; D014357:Trypsin; D015032:Zinc