Protein Purification And Crystallographic Studies Of The Orphan Nuclear Receptor ERR?

Nuclear receptors(NRs),important members of transcriptional factors,are constituted by classic receptors,adopted-orphan receptors and orphan receptors.Classic receptors bind to endogenous ligands,such as steroid hormones,thyroid hormones and vitamin A derivatives.Transcriptional activity of the NRs is regulated by these endogenous ligands respectively.Estrogen related receptors(ERRs)include three subtypes of ?,? and ?(ERR?,ERR? and ERR?),which are associated with many biological processes.ERRs,members of orphan nuclear receptor subfamily,are homologous to the estrogen receptors(ERs)in structure,but are not activated by the estrogens.Subsequent studies indicate that a series of synthetic compounds are the ligands of ERRs,such as 4-OHT,DES and BPA.The ERR subfamily is termed as an"adopted orphan receptor".ERR? plays an essential role in embryonic development,cell replication and differentiation.In ERR? knock-out mice model,chorion formation is hampered accompanied by the increase of trophoblast giant cells which eventually lead to a fetal death.In a similar structure with canonical NRs,ERRs have four typical domains.The Ligand binding domain(LBD)and DNA binding domain(DBD)are highly conserved among the three ERRs subtypes.ERRs exhibit constitutively transcriptional activity in a ligand independent manner.The structures of ERRa LBD and ERRy LBD reveal the molecule mechanism of constitutive activation.The aromatic side chains in the LBD occupy the ligand binding pocket(LBP),so ERR?only permits to accommodate the small molecules in low molecular weight.Further studies uncover that BPA-binding can not interfere with the coactivators recruitment of ERRy LBD.And the structure of apo ERRa LBD with PGC-1? is also in a constitutive activation state,because the side chain F328 in Helix 3 fills up the LBP and acts as an "internal" ligand.The PGC-1? adopts an inverted LXXLL motif(LLKYL motif)to interact with AF-2.As so far,the structural information of ERR? LBD is still ambiguous.In fact,lacking of the ERR? LBD structure may be attributed by the protein stability and solubility.We have constructed a mutation 1 in ERR? LBD in order to enhance the stability of the protein for purification and crystallization.Compared with the wild-type,the transcriptional activity of the mutant ERR? has no significant difference verified by the cell-based reporter assays.In our study,we identified a novel ligand H3A11 for ERR? and have obtained crystals of six different ERR?complexes,providing a solid foundation for the further structural studies.These crystal structures will give a better understanding of the transcriptional regulation of ERR? and provide a drug-design template for ERR? relevant disease treatment.