Characterization of bacterially expressed rat estrogen receptor β ligand binding domain by mass spectrometry: Structural comparison with estrogen receptor α

Functional rat estrogen receptor β ligand binding domain (rERβ LBD, aa 210–485) and human estrogen receptor α ligand binding domain (hERα LBD, aa 301–553) were expressed in Escherichia coli. Hormone binding assays revealed that both ERβ and ERα LBDs bound the natural ligand estradiol (E2) with similar affinity (Kd ∼ 100 pM). Competitive binding experiments were carried out with ICI 164384, 4-hydroxytamoxifen, 16α-bromo-estradiol, and genistein employing [3H]E2 as a tracer. No significant differences in responses of ERα and ERβ LBDs to ICI 164384 and 4-hydroxytamoxifen were observed. 16α-Bromo-estradiol and genistein discriminated between the ER subtypes and acted as ERα and ERβ selective ligands, respectively. Final purification of recombinant proteins was achieved on an E2 affinity column, where they were subjected to in situ carboxymethylation. The partially carboxymethylated proteins actively bound E2. The carboxymethylated rERβ LBD had a molecular mass of 32251.6 Da, equivalent to the calculated mass with the addition of three carboxymethyl groups. No other proteins (of lower or higher molecular mass) were detected, so the LBD was considered structurally authentic and pure. By using a combination of intact protein mass spectrometric fragmentation and trypsin proteolysis (98% sequence coverage), it was established that rERβ cysteine-289 and -354 were not carboxymethylated on the affinity column, suggesting that they were shielded from alkylation in the E2-bound conformation state. Concurrent analysis of hERα LBD showed that under the same experimental conditions, the two equivalent ERα cysteines were not alkylated (αC381 and αC447). These data support close structural relationship between the E2-bound ERα LBD and ERβ LBD proteins.