Purification and biochemical characterization of mutant ligand binding domain of human estrogen receptor α protein

Area of Science:

• Endocrinology
• Molecular Biology
• Oncology

Background:

• Estrogen receptor alpha (ERα) gene (ESR1) mutations are a primary cause of endocrine therapy resistance in breast cancer.
• ESR1 mutations, particularly Y537S and D538G, lead to estrogen-independent receptor activity and reduced sensitivity to existing treatments.
• These mutations are common in metastatic ER-positive breast cancer, correlating with poorer patient survival.

Purpose of the Study:

• To express and purify the ligand-binding domains (LBDs) of wild-type, Y537S, and D538G human ERα.
• To biochemically characterize these purified ERα LBDs to confirm their activity and stability.
• To establish a foundation for screening small molecules that can differentiate between wild-type and mutant ERα.

Main Methods:

• Bacterial expression and purification of wild-type, Y537S, and D538G human ERα LBDs.
• Biochemical assays including homogeneous time-resolved fluorescence and polar screen ERα competition assays.
• Thermal shift assays to assess protein stability.

Main Results:

• Purified ERα LBDs (wild-type, Y537S, D538G) demonstrated biochemical activities consistent with full-length proteins.
• Wild-type ERα LBD required estradiol for coactivator binding, while Y537S and D538G mutants bound coactivators spontaneously.
• The Y537S mutant exhibited higher affinity for coactivator peptides compared to D538G, and mutations conferred increased domain stability.

Conclusions:

• The purified ERα LBDs accurately model the behavior of full-length mutant receptors.
• These purified domains are suitable for developing screening assays to identify drugs targeting specific ERα mutations.
• This work provides a valuable tool for advancing the treatment of endocrine therapy-resistant breast cancer.