NFkappaB selectivity of estrogen receptor ligands revealed by comparative crystallographic analyses

Abstract

Our understanding of how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches. However, progress has been hampered by 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 that are common to steroid hormone receptors are circumvented by mutations that stabilize well-characterized conformations of the receptor. We use this approach to present the structure of an apo steroid receptor that reveals 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 NFkappaB-selective signaling through the estrogen receptor, thus 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 structural features of signaling specificity that would not be apparent in a single structure.

Fig. 1. Overall Structure of the Tyr-537-Ser ERα/Grip1 complex

(a) Cartoon of ER, showing domain boundaries and names, and abbreviations. (b) The Tyr-537-Ser ERα mutant or the Wt ERα LBD were incubated with excess tritiated estradiol, bound to glass beads, and the unbound ligand washed away. Picomoles of bound ligand were calculated, and compared with the numbers of picomoles of the receptor, to determine % of ligand bound. Shown is mean + SEM. (c) The secondary structure is shown as ribbon diagram, with the GRIP1 peptide colored red, the helices colored blue, and the beta sheet colored yellow. (d) The alpha carbon trace of the mutant ER (orange) was superimposed on the DES/ERα structure (blue) using all the backbone atoms. The green boxes denote regions of significant difference in secondary structure.

Fig 2. Identification of a mutation that stabilizes the antagonist conformation of ERα

(a) Shown is the structure of tamoxifen-bound ERα (PDB:3ERT) as a ribbon diagram. Helices 3–5 are colored pink, and helix 12 is colored red. (b) Molecular modeling suggests that the mutation Leu-536-Ser of ERα promotes a stabilizing interaction between helix 12 and Glu-380 in helix 3.

Fig 3. A solvent-accessible channel in the Tyr-537-Ser ERα LBD

(a) The structure of genistein-bound ERα Tyr-537-Ser is depicted as ribbon diagram, showing only a portion of the molecule that interacts with the ligand. The closed interface between helix 11 and L7-8 is shown by a red circle. (b) The mutant ERα structure with no added ligand is rendered as in panel A, with the red circle highlighting the altered conformation of His-524, and the solvent accessible channel. (c) The apo and genistein-bound ERα Tyr-537-Ser structures were superimposed over the backbone residues. A surface rendering of the apo structure was colored by electrostatic potential, revealing the solvent accessible channel and the superimposed genistein molecule as a stick rendering.

Fig 4. Comparison of ERα wild type and Tyr-537-Ser crystal structures

(a) The wild type Tyr-537 ERα is shown from the structure of genistein-bound ERα (PDB code:1X7R). Helices 3, 11, and 12 are shown as ribbons, while the loop between helices 11–12 is rendered as a stick figure. This illustrates the hydrogen bond formed with Asn-348, and the location of Leu-536 in a solvent exposed position. (b) The mutant Tyr-537-Ser ER bound to genistein forms a hydrogen bond with Asp-351, allowing the rotation of Leu-536 out of the solvent. (c) The amino acids lining the pocket are shown for the mutant and wild-type ERα (PDB code:1X7R) bound to genistein. The two structures were superimposed using the backbone atoms of amino acids within 4.2 Å of the ligands. The Tyr-537-Ser mutant ERα with genistein is colored gray, with the corresponding amino acids colored green. The wild-type ERα structure is colored orange. A structurally conserved water molecule is also shown.

Fig 5. Transcriptional activity of NF-κB selective ER ligands

(a) MCF-7 cells were transfected with a 3×ERE-luciferase reporter. The next day, cells treated for 24 hrs with the indicated ligands, and processed for luciferase activity. Shown is mean + SEM from 4–8 wells for each dose. (b) MCF-7 cells were transfected with a 5×NFκB-luciferase reporter. The next day, cells were treated for 6 hrs with TNFα and the indicated ligands, and then processed for luciferase activity. (c) MCF-7 cells were treated for 2 hrs with TNFα and the indicated ligands, and then processed for RT-qPCR. The mRNA for IL-6, IL-8, or MCP-1 was normalized to 18S mRNA. Shown is the mean + SEM for duplicate measurements.

Fig 6. Crystal structures of ER bound to NFκB selective compounds

(a) The structure of the oxabicyclic diarlyethylene compound/ERα LBD is shown as a ribbon diagram, with the ligand and selected residues in the ligand binding pocket shown as sticks. (b) The structures of ERα Tyr-537-Ser bound to the indicated ligands were each superimposed with the structure bound to the full agonist, ether estradiol compound (38), using all of the main chain atoms. Shown are selected residues in the pocket, and alpha carbon traces for helices 11 and 12. The ether estradiol bound structure is colored green (ligand not shown), and the NFκB selective structures, and compounds, are colored gray. The red arrows denote the shifts in helix 11 induced by the NFκB selective compounds.

Fig 7. Structural and Biological Characterization of an Intermediate Agonist

(a–b) Luciferase activity was assayed as described in Fig 5. (c) The structure ERα Tyr-537-Ser bound to the oxabicycic diarylmethylene was superimposed with the full agonist, ether estradiol structure, as described in Fig 6.