Herein we presented the crystal structure of CPA bound to the T877A AR LBD. Based on the structural findings observed in the T877A·CPA complex, we predicted that the L701A AR mutant would increase agonist activity of CPA due to the relief of the strain on helix 3 and recovery of the packing of the loop between helices 11 and 12 into the groove observed in other AR LBD structures. As predicted, CPA stimulated transcriptional activation in the L701A AR similar to T877A AR. Conversely, HF exhibited no response in the L701A AR, demonstrating the ability of this mutation to selectively confer agonist activity to CPA. This result further suggests that the gain in AR function observed in the L701A AR is due to the interaction with CPA, because HF does not alter the position of Leu-701 in the T877A AR LBD (8
) relative to the published WT·DHT structures (16
). Electron density maps for AR residues 886–890 in the T877A·CPA structure demonstrated that this region is not well ordered. The flexibility of this loop, however, provides insight to understanding the dynamics of the AR LBD. Unlike other published AR LBD complexes, the T877A·CPA structure elucidates a gateway for the ligand to enter the binding pocket. Crystal structures of the estrogen receptor α
LBD have shown that the dynamic properties of helix 12 act as a lid that provides an entrance for ligands to reach the binding cavity (9
). However, the C terminus of the AR contains a β
-sheet that may restrict the mobility of helix 12 relative to the crystal structures of the estrogen receptor α
, which were determined with a truncated C terminus. Furthermore, evidence of the alteration in the folding of C terminus of helix 11 and adjacent loop region in the T877A·CPA structure suggests that this dynamic portion of the AR may provide ligand accessibility to the binding pocket. As seen from , it can be anticipated that CPA binding in the presence of the Thr-877 side chain would push helix 11 away from the binding pocket and thus intensify the unfolding of the AR LBD that already occurs in this region. Such an alteration to the AR LBD structure would resemble the change induced by the ligand, GC-24, in the human thyroid hormone receptor β
). Transcriptional activation data suggest that both the T877A and L701A AR relieve a sufficient amount of strain when complexed to CPA to recover functional AF-2 formation as seen from the ability of CPA to induce transcriptional activation at a low concentration (i.e.
). Conversely, bicalutamide likely unfolds the AR LBD at the N terminus of helix 12 from steric interaction with Met-895 as shown from our previous report in which W741L and M895T convert it to an agonist by reducing the bulk in this region of the AR (8
Although CPA acts as an antagonist in the GR similar to the AR, it is an agonist ligand for the PR. The corresponding residue to Leu-701 in the GR and PR were mutated to decrease bulk in the region presumably occupied by the 17α
-acetate of CPA upon binding to these other steroid receptors. The M560A mutant in the GR predictably converted CPA from an antagonist in the WT (22
) into a potent agonist. This result is explained by the ability of the GR to accommodate the 17α
-acetate of CPA and prevent disruption of the GR-LBD folding of the loop linking helices 11 and 12. The M560A mutant severely weakened the activity of dexamethasone, which is likely attributed to loss of van der Waals contacts. Both CPA and progesterone displayed potent activity in the WT PR eliciting transcriptional activation at low nanomolar concentrations as expected. However, almost complete loss of activity was observed with progesterone in the L715A mutant with preservation of CPA activity.
The expanded binding pocket seen in the AR T877A·CPA complex demonstrates an expanded binding pocket not seen in other reported AR LBD crystal structures to date. This finding explains the discrepancy observed in docking experiments and the incompatibility of CPA with the ligand-binding pocket in the AR from the WT·DHT and T877A·DHT complexes (16
). The receptor interactions in this cavity provide information useful for structure-based drug design. Ligands that hydrogen bond to Ser-778 will likely offer a means of optimizing binding affinities of new compounds that target the AR. Unfortunately, receptor flexibility is difficult to predict with molecular modeling. Docking software such as FlexX and AutoDock employ rigid protein structures, which limits the ability to accurately predict binding conformations of AR ligands (10
). Here we demonstrate the use of ligand-receptor co-crystallography to determine flexible regions of the AR and identify new insight for structure-based drug design and receptor function.