Structural and biochemical analysis of the Estrogen-Related Receptor α and complex with TMPRSS2 promoter DNA

In TMPRSS2 fusion-positive prostate cancer, ERRα is involved in regulation of ERG and promotes the androgen receptor independent signaling in the cancer progression. The ERRα binds to the ERREs (estrogen-related receptor response elements) present at −5042 bp of the TMPRSS2-promoter and enhances the ERG overexpression that causes prostate cancer progression. To dissect the structural basis of the ERRα recognition to the TMPRSS2 promoter DNA, we have purified the full-length ERRα (ERRαFL), NTD deleted construct (ERRαΔNTD), and the DNA-binding domain (ERRαDBD) proteins and performed the binding analysis with 30 bp TMPRSS2-promoter DNA (5′ -AGTCCAAGGTCGGTGGATC ACAAGGTCAGG-3′). Circular dichroism analysis showed that all three ERRα proteins adopt native secondary structures. DNA binding induced subtle changes in the secondary structures, while enhancing the thermal stability (Tm) of all ERRα proteins. Binding analysis showed that ERRαDBD bound weakly to the DNA, whereas ERRαFL and ERRαΔNTD exhibited substantially higher affinities ∼120-fold and ∼131-fold than ERRαDBD, respectively. Small-angle X-ray scattering (SAXS) analyses revealed a dimeric ERRαFL structure and an ERRαFL-DNA complex (2:1) structure in solution and fitted well with Alpha Fold model of apo and DNA bound complex of ERRαFL. Furthermore, 100 ns dynamics simulations on apo and DNA-bound ERRα proteins showed that all proteins remained structurally stable, with flexibility largely confined to loop regions of ERRα proteins. Our biophysical, DNA binding and structural analyses have revealed the mechanism involved in ERRα recognition of the TMPRSS2-promoter DNA, which provides insight into ERRα-mediated transcriptional regulation and development of anticancer drugs against ERRα-driven prostate cancer.