Expression, purification and crystallization of the ancestral androgen receptor–DHT complex

Abstract

Steroid receptors (SRs) are a closely related family of ligand-dependent nuclear receptors that mediate the transcription of genes critical for development, reproduction and immunity. SR dysregulation has been implicated in cancer, inflammatory diseases and metabolic disorders. SRs bind their cognate hormone ligand with exquisite specificity, offering a unique system to study the evolution of molecular recognition. The SR family evolved from an estrogen-sensitive ancestor and diverged to become sensitive to progestagens, corticoids and, most recently, androgens. To understand the structural mechanisms driving the evolution of androgen responsiveness, the ancestral androgen receptor (ancAR1) was crystallized in complex with 5-dihydrotestosterone (DHT) and a fragment of the transcriptional mediator/intermediary factor 2 (Tif2). Crystals diffracted to 2.1 Å resolution and the resulting structure will permit a direct comparison with its progestagen-sensitive ancestor, ancestral steroid receptor 2 (AncSR2).

Evolutionary tuning of a key helix drove androgen selectivity

“…The AR1-DHT-Tif2 ternary complex was crystallized as previously described [17]. Coordinates and structure factors were deposited in the Protein Data Bank (PDB) with the accession number 7RAE.…”

“…Nineteen systems were prepared for molecular dynamics simulations using the crystal structures of the ligand binding domains of AncAR1-DHT [17] and AncAR1-rev-Progesterone, and AncSR2-progesterone (PDB 4LTW) as starting structures. The Tif2 peptide was excluded from all complexes for MD.…”

“…Consistent with its identity as the ancestor of all androgen-activated steroid receptors, AncAR1 is activated by the androgen DHT but not by progesterone, which differs only by its C17 substituent (Figure 2A). To understand how AncAR1 interacts with androgens, we determined the x-ray crystal structure of the AncAR1 ligand binding domain (LBD) in complex with DHT and a fragment of the TIF2 coregulator protein to 2.1 Å [17](Figure 2B, S1). The structure adopts the canonical "alpha helical sandwich" conformation observed for all steroid receptor LBDs.…”