Small-angle X-ray scattering studies on structures of an estrogen-related receptor alpha ligand binding domain and its complexes with ligands and coactivators

Abstract

We have investigated the structure of the mouse estrogen-related receptor alpha ligand binding domain (mERR alpha LBD) with a flexible hinge region (including more than 30 residues) in solution and the variations of its structure in response to binding with various ligands and coactivators by using synchrotron small-angle X-ray scattering (SAXS) and biochemical analysis. The mERR alpha LBD alone and in complex with the domain containing all three leucine-rich repeats of human peroxisome proliferator-activated receptor gamma coactivator-1 alpha (hPGC-1 alpha) were both cloned and expressed in Escherichia coli and purified to homogeneity. We also synthesized steroid receptor coactivator-1 (SRC-1), a 15-mer peptide corresponding to the leucine-rich repeat 4 of human SRC-1. We tested 4-hydroxytamoxifen, diethylstilbestrol, chlordane, genistein, daidzein, and biochanin A as ligands. SAXS and biochemical analyses were used to show that the apo mERR alpha LBD is present as a homodimer in solution. The apo mERR alpha LBD homodimer has an asymmetrically ellipsoidal shape with a lower region that includes the extended hinge domain. It was found that the homodimer always undergoes some structural changes in cooperation with the hinge domain when it binds with ligands. These structural changes are probably due to changes in the conformation of the mERR alpha LBD ligand binding pocket, which has a relatively small volume when ligand bindings occur. Overall, all the tested ligands have similar impacts on the structure of the mERR alpha LBD. In contrast, the mERR alpha LBD:hPGC-1 alpha complex has a Y-shaped structure, which is quite different from the structures of the mERR alpha LBD bound with ligands. This result provides important information about the spatial orientation of the flexible disordered and/or relatively rigid regions bearing all three L1, L2, and L3 motifs of the partially unfolded hPGC-1 alpha fragments, which are involved in the interaction with the mERR alpha LBD. The mERR alpha LBD was found to have a strong binding affinity with the hPGC-alpha coactivator fragment, which arises from the cooperativity of the three leucine-rich motifs in the coactivator binding mode. In contrast, the mERR alpha LBD complex with SRC-1 was found to have a structure very similar to that of the apo mERR alpha LBD, which provides a foundation for a ligand-independent understanding of the constitutively transcriptional activity of apo ERRs.