Study Of Interaction Between Retinoic Acid Receptor RXRα And Transcriptional Repressor TGIF1

Retinoid X receptor RXRαis a member of the nuclear receptor（NR）protein family and has attracted much attention as a drug target.RXRαbinds to retinoic acid（RA）through its ligand binding domain（LBD）,and then interacts with coactivators such as SRC2 to activate gene expression.TGIF1 is a transcription repressor of the homeodomain（HD）family,which can regulate RA and other signaling pathways.In most cases,TGIF1uses its HD to bind to TGTCA elements in gene promoters to inhibit gene expression,but this element is often absent in promoters of RA-responsive genes.In this case,TGIF1regulates RA responsive genes through the interaction of HD with RXRαLBD as a co-repressor,but the molecular mechanism of RXRα-LBD binding to TGIF1-HD is still unclear.Existing studies on the interaction between RXRα-LBD and its cofactor proteins from the perspective of structure are mainly crystallographic studies.Due to the fact that RXRα-LBD exists in solution as a mixture of monomer,dimer and tetramer,so it is difficult to obtain uniform monodisperse samples and lack of study in solution.Moreover,the molecular weights of dimer and tetramer are high.These issues hinder the study using methods such as NMR and small-angle X-ray scattering.In this paper,the known structures of RXRα-LBD homodimer was analyzed,and two residues D379 and L420 on the dimerization interface were selected.Three RXRα-LBD mutants were designed and constructed toward the selected residues of D379 and L420,and high purity protein samples of the three were prepared.Circular dichroism analysis showed that the structure of the three mutants did not change significantly compared with that of the wild type.Size exclusion chromatography and analytical ultrafast centrifugation（AUC）experiments showed that the oligomeric forms of the three mutants changed to different degrees,and the dimer proportion decreased.Therein,the RXRα-LBD^D379A-L420Gwith two mutated residues showed a uniform monomer form in solution.Fluorescence quenching and Pull-down experiments demonstrated that the binding ability of the three RXRα-LBD mutants to ligand 9-cis-RA and cofactors（SMRT and SRC2）did not change compared with the wild type.Pull-down experiment proved that L420mutation reduced the stability of the heterodimer formed by RXRα-LBD and RARα-LBD,while D379 mutation had no effect on this.Through analyzing the known heterodimer structures of RXRα-LBD and other NRs,it was found that D379 and L420 played different roles in the formation of heterodimers.L420 is involved in the formation of all heterodimers with known structures,while D379 has little interaction with RARα/β,LXRα/β,and THRα/β.The interaction mechanism between RXRα-LBD and TGIF1-HD was preliminarily studied by using the RXRα-LBD protein samples in the form of monomers in solution.The interaction between TGIF1-HD and RXRα-LBD in solution was verified by NMR,and the dissociation constant was determined to be about 47μM.The dissociation constants of wild-type RXRα-LBD and RXRα-LBD^D379A-L420Gmutant to TGIF1-HD were further determined by ELISA.The results were consistent with the results of NMR titration,and the dissociation constants of wild-type and mutant to TGIF1-HD were basically the same.Wild-type RXRα-LBD and RXRα-LBD^D379A-L420Gmutant formed complexes with TGIF1-HD at a stoichiometric ratio of 1:1 as demonstrated by AUC experiments.It has been proved by ELISA and Pull-down experiments that ligand 9-cis-RA and corepressor SMRT can inhibit the binding of RXRα-LBD to TGIF1-HD,indicating that the binding site of TGIF1-HD in RXRα-LBD is similar to those of 9-cis-RA and SMRT.In addition,DNA containing TGTCA element can inhibit the binding between TGIF1-HD and RXRα-LBD as revealed by Pull-down experiments,indicating that TGIF1-HD interacts with RXRα-LBD by using sites close to those for binding to DNA.This article preliminarily explained the interaction mechanism between RXRα-LBD and TGIF1-HD,which laid the foundation for obtaining the structure of the complex between the two and fully elucidting the interaction mechanism.