Structure Of Plant Co-repressor TPL/TPR Protein Provides Insights Into Mechanism Of EAR Motif Binding

TOPLESS(TPL) and TOPLESS-related(TPR) proteins comprise a conserved superfamily of plant transcriptional co-repressors, for short, TPL/TPR family. TPL/TPR proteins are related to Tup1, Groucho, TLE((transducin-like enhancer of split) co-repressors in yeast, insects, and animals. In plants, through interaction with small ethylene response factor – associated amphiphilic repression(EAR) motifs found in diverse transcriptional repressors, TPL/TPR proteins regulate a wide spectrum of plant physiology like development, stress responses, and hormone signaling. For a long time, people is interested in the mechanism how co-repressor TPL/TPR proteins interact with the repressor EAR motif which remains unknown.We confirm the conserved N terminal of co-repressor TPL/TPR family proteins, which we call it TOPLESS domain(TPD), interacting with the EAR motif. In order to understand the structural basis of this interaction, we determined the crystal structures of the TPD of rice(Os) TPR2 in apo(apo protein) state and in complexes with the EAR motifs from Arabidopsis NINJA(novel interactor of JAZ), IAA1(auxin-responsive protein 1), and IAA10, key transcriptional repressors involved in jasmonate and auxin signaling. The Os TPR2 TPD adopts a new fold of nine helices, followed by a zinc finger, which are arranged into a disc-like tetramer. The EAR motifs in the three different complexes adopt a similar extended conformation with the hydrophobic residues fitting into the same surface groove of each Os TPR2 monomer. Sequence alignments and structure-based mutagenesis indicate that this mode of corepressor binding is highly conserved in a large set of transcriptional repressors, thus providing a general mechanism for gene repression mediated by the TPL family of corepressors.D53 protein is a repressor in the Strigolactones(SL) signaling pathway. D53 binds to general corepressors of the TPL/TPR family proteins which have essential roles in plant development and hormone signaling. It is anticipated that there are three classical Lx Lx L EAR motif, which olny one EAR motif shows obvious binding to the TPL/TPR TPD protein, and we term it D53 peptide. We present the crystal structure of the complex of this motif bound to the TOPLESS domain(TPD) of rice TPR2. The motif binds to the same binding groove of each TPD monomer as EAR motif peptides involved in jasmonate and auxin signaling, but makes an additional interaction with a second site in the vicinity of the TPD zinc finger to mediate TPD tetramer-tetramer interaction. We extensively validated the two binding sites by mutational and structural analysis and demonstrate biochemically a D53 EAR-mediated formation of TPD oligomeric assemblies. Moreover, we demonstrate that the TPD directly binds nucleosomes and the tails of histones H3 and H4, and that D53 peptide-induced formation of higher order TPD complexes markedly stabilizes the nucleosome-D53 interaction. Together, these results establish a new TPD-repressor binding mode that stabilizes TPD-nucleosome complex formation through induced TPD oligomerization, and that may bring the putative D53 remodeling activity to TPD-bound nucleosomes.