NFκB selectivity of estrogen receptor ligands revealed by comparative crystallographic analyses

Abstract

Understanding how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches. However, progress has been hampered by significant misfolding of the ligand binding domains in heterologous expression systems and by conformational flexibility that interferes with crystallization. Here, we show that protein folding problems common to steroid hormone receptors are circumvented by a mutations that stabilize well-characterized conformations of the receptor. We use this approach to present the first structure of an apo steroid receptor, revealing a ligand-accessible channel, allowing soaking of preformed crystals. Furthermore, crystallization of different pharmacological classes of compounds allowed us to define the structural basis of NFκB selective signaling through ER, revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression. The ability to crystallize many receptor-ligand complexes with distinct pharmacophores allows one to define the structural features of signaling specificity that would not be apparent in a single structure.

Structural and mechanistic insights into bisphenols action provide guidelines for risk assessment and discovery of bisphenol A substitutes

“…S8 A and B). These data complement other comparisons that were made earlier with a nonsteroidal ligand (34,35). Additional characterization of the Y537S mutant via transient transfection of HeLa cells, Thermofluor and fluorescence anisotropy indicated that the mutation stabilizes the active conformation of the receptor without modifying the relative potencies of compounds (SI Appendix, Figs.…”

“…4A). These suboptimal or complete lack of interactions induce a substantial reorientation of the H524 imidazole ring, which, as previously reported by Nettles et al (34), perturbs a key hydrogen bond network involving residues from loop L6-7, H3, and H11. Last but not least, we observed that, in the E 2 -bound structure, the 18-methyl group of E 2 is in contact with L525 and imposes a conformation that strengthens a network of van der Waals interactions involving T347, L525, and L536 (Fig.…”

“…We next considered how the two binding modes of bisphenols may contribute to the destabilization of the AF-2 surface. By comparing our four structures, we observed that previously recognized ligand-H11 stabilizing interactions observed in the E 2 -bound structure are altered in the bisphenol-containing complexes (34,35). For example, E 2 makes an important stabilizing interaction with G521 (3.95 Å), which is severely weakened in the structures with bisphenols in the "BPA-like" conformation (4.65 Å) or completely abolished in the structures with bisphenols in the "BPC-like" conformation (>5.20 Å; Fig.…”

“…In an attempt to gain structural insight into the binding mode of BPA to ERs, we subjected the WT ERα LBD complexed with BPA to crystallization assays. After several rounds of unsuccessful trials, we used the recently reported ERα LBD mutant (Y537S), which has been shown to stabilize the agonist-bound conformation of ERα, and, in turn, facilitate crystallization of weak agonists (34). To ensure that this mutation at the surface of the protein will not compromise the accuracy of our structural analysis, we cocrystallized ERα-Y537S LBD with E 2 and SRC-1 NR2.…”

Implications of the binding of tamoxifen to the coactivator recognition site of the estrogen receptor

“…This is supported by the notion that the binding of HT to the AF-2 surface is of lower affinity compared with the cognate ligand-binding pocket (Wang et al 2006). Recently, it was reported that the Y537S ERa LBD mutation allows for the conformational stabilization during protein purification and subsequently allows for the addition of ligands to purified apoprotein (Nettles et al 2008). …”