Characterization of hsp90-binding to the the PASB domain of the Ah receptor

The Ah receptor (AhR) is a ligand-dependent transcription factor, which regulates the biochemical and toxic effects of structurally diverse chemicals. Hsp90, one subunit of the AhR complex, appears to direct proper folding and maintenance of the high affinity ligand binding conformation of the AhR in some species. Using a structural homology model we developed for the ligand- and hsp90-binding PASB domain of the AhR to guide our mutagenesis, we have examined the physical interactions of hsp90 with the AhR and subsequent effects on AhR functionality (hsp90, ligand and DNA binding and transcriptional activation). Deletion of the PASB domain resulted in the complete loss of hsp90-binding and constitutive ligand-independent activation of DNA-binding and reporter gene transcription, suggesting that documented interactions of hsp90 with the AhR bHLH domain are insufficient on their own for hsp90 binding. Deletions within the AhR PASB domain resulted in intermediate levels of hsp90 binding and revealed that the bulk of the central PASB 5 strand ß-sheet is required for the optimal hsp90-binding. PASB deletions that eliminated amino acids 339-362 resulted in ligand-independent transformation and DNA-binding, similar to full PASB deletion. Based on the AhR PASB model, these results indicate that two ß-strands (Gß and Hß ) along with the interconnecting flexible loop are essential for ligand-induced activation of AhR transformation possibly by weakening of the hsp90-AhR interactions within this region. (NIH ES07685)