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 5α-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).

Keywords: 5α-dihydrotestosterone; ancestral androgen receptor; steroid receptors.

Figure 1

Following a series of affinity columns, ancAR1–DHT was purified to homogeneity. Lane 1, TEV protease-cleaved ancAR1-MBP fusion protein. Lane 2, purified ancAR1. Lane 3, cleaved MBP. Lane M contains molecular-weight markers (labeled in kDa).

Figure 2

Crystals of ancAR1–DHT. The long rod-shaped crystals are approximately 100–200 µm in length. The crystals grew in 20% PEG 1000, 0.3 M MES pH 6.5.

Figure 3

Diffraction image of an ancAR1–DHT crystal. The detector edge corresponds to 2.15 Å resolution.