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

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+ 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.

Design of Affinity Tags for One‐Step Protein Purification from Immobilized Zinc Columns

“…As discussed above, Figure 4 shows that His 6 -rGFPuv elutes from the column at pH 5.0. Elution at pH 5.0 may lead to leakage of toxic zinc (21). Additionally, many proteins exhibit decreased stability at pH 5.0.…”

“…Chromatography column C 10/20 (Pharmacia Biotech, Uppsala, Sweden) was packed with 1 mL of Chelating Sepharose Fast Flow 6B (Pharmacia Biotech, Uppsla, Sweden). Ten column volumes (CV) of deionized water was passed through the column followed by 10 CV of either 50 mM ZnCl, 50mM CuSO 4 , or 50 mM NiCl 2 ‚6H 2 O. Unbound Cu 2+ and Ni 2+ were removed using 10 CV of 0.1 M sodium acetate, 0.5 M NaCl (pH 4.0) buffer, and unbound Zn 2+ was removed using 10 CV of 20 mM Na 2 -HPO 4 /NaH 2 PO 4 , 0.5 M NaCl (pH 6.0) to prevent the possible removal of bound zinc ions (21). The column was then equilibrated with 10 CV of the elution buffer at pH 7.5.…”

“…This pattern of elution implies that directing the release of a target protein to the region between pH 6.0 and 7.0 would lead to an extremely high degree of purification. Elution between pH 6 and 7 will also ensure that there is no zinc leakage (21). Finally, the relatively high elution pH in the range of pH 6-7 will ensure better protein stability than elution at lower pH values.…”

Immobilization and affinity purification of recombinant proteins using histidine peptide fusions

“…This metal ion was chosen due to its relatively low binding to native proteins, as compared to Ni2' or Cu2+ [36]. Native staphylococcal protein A has previously been demonstrated not to bind Zn2…”

The purification of TEM-?-lactamase from cell extract using immobilized copper affinity chromatography

“…Several different metal ions may be employed, including copper, nickel, and zinc (Hutchens and Li, 1988).…”