Surveying helix 12 dynamics within constitutively active estrogen receptors using bipartite tetracysteine display

Abstract

Somatic Y537S and D538G mutations within the estrogen receptor alpha ligand-binding domain (ERα-LBD) have been linked to enhanced cell proliferation, survival, and metastases in ER-positive breast cancers. Such mutations are thought to confer ligand-independent receptor activation by increasing the flexibility of helix 12 (H12), a segment within the ERα-LBD that acts as a dynamic regulator of ERα activity. We employed bipartite tetracysteine display coupled with the biarsenical profluorophore FlAsH-EDT₂ to monitor ligand-independent structural transitions of H12 in ERα-LBDs that include Y537S or D538G mutations. Our results show that in the absence of 17β-estradiol, Y537S and D538G mutations cause H12 to fold into a "stable agonist" conformation that is similar to liganded (17β-estradiol-bound) wildtype ERα-LBDs. We also observed that stable agonist conformations adopted by unliganded Y537S or D538G mutants resist H12 transitions to inactive states. Taken together, these results indicate that Y537S and D538G mutations endow constitutive activity to the ERα by directly influencing H12 dynamics. Furthermore, our findings provide insight into how Y537S and D538G mutations impart resistance to endocrine or antiestrogen therapies in ER-positive breast cancers.

Keywords: 17β-estradiol; biarsenical profluorophore; bipartite tetracysteine display; estrogen receptor alpha; helix 12 transitions; protein dynamics; type 1 nuclear receptor.