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

The acquisition of mutations in the estrogen receptor alpha (ER alpha) gene (ESR1) is a key driver in the development of resistance to current endocrine therapy in breast cancer. Clinical studies have shown that ESR1 mutations are frequently observed in patients with metastatic ER-positive breast cancer and are associated with poor survival. Activating ESR1 somatic mutations, particularly Y537S and D538G, drive estrogen-independent activities in cellbased studies and these mutant receptors are less sensitive to current endocrine therapies. Here, we describe the bacterial expression and purification of the ligand binding domains of wild-type, Y537S, and D538G human ER alpha proteins. The biochemical activities of these domains were confirmed by homogeneous time-resolved fluorescence and polar screen ER alpha competition assays. The wild-type domain binds to coactivator peptides only in the presence of the ligand estradiol, whereas the Y537S or D538G domains bind coactivator peptides spontaneously even without estradiol, with the Y537S domain showing higher affinity. Thermal shift assays showed that the mutations stabilized these domains. Our purified human ER alpha wild-type, Y537S, and D538G ligand binding domains recapitulate the biological activities ascribed to the full-length proteins and can therefore be used for small molecule screens that seek to discriminate between wild-type and mutant ER alpha.