Structure of the Arabidopsis TOPLESS corepressor provides insight into the evolution of transcriptional repression

Abstract

Transcriptional repression involves a class of proteins called corepressors that link transcription factors to chromatin remodeling complexes. In plants such as Arabidopsis thaliana, the most prominent corepressor is TOPLESS (TPL), which plays a key role in hormone signaling and development. Here we present the crystallographic structure of the Arabidopsis TPL N-terminal region comprising the LisH and CTLH (C-terminal to LisH) domains and a newly identified third region, which corresponds to a CRA domain. Comparing the structure of TPL with the mammalian TBL1, which shares a similar domain structure and performs a parallel corepressor function, revealed that the plant TPLs have evolved a new tetramerization interface and unique and highly conserved surface for interaction with repressors. Using site-directed mutagenesis, we validated those surfaces in vitro and in vivo and showed that TPL tetramerization and repressor binding are interdependent. Our results illustrate how evolution used a common set of protein domains to create a diversity of corepressors, achieving similar properties with different molecular solutions.

Keywords: TOPLESS; auxin signaling; corepressor; crystal structure; tetramerization.

Fig. 1.

TPL N terminus structure and conservation. (A) AtTPL protein schematic representation. (B) Cartoon representation of AtTPL184 homodimer. LisH, CTLH, and CRA domains are colored in red, gold, and blue in one of the two monomers, respectively. (C) TBL1 (green) and AtTPL184 (blue) structure superimposition. (D) Sequence alignment of the N terminus of TPL and A. thaliana (Ath) TPR proteins. Atr, Amborella trichopoda; Osa, Oryza sativa; Mpo, Marchantia polymorpha; Ppa, Physcomitrella patens; Nae, Nothoceros aenigmaticus; Kfl, K. flaccidum. Triangle, amino acids involved in the interaction with the IAA peptide; star, amino acids involved in tetramerization; circle, N176 residue.

Fig. 2.

TPL tetramerization. (A) AtTPL184 tetrameric form I (dimers in blue and yellow). (Inset) Close-up view of the tetramerization interface with a rotation of about 90° to better view interacting residues. The interactions between chains C and D (Left) are slightly different from those between A and B (Right), as indicated in SI Appendix, Table S3. Residues mutated in different experiments are shown in red. (B) AtTPL184 tetrameric form II and close-up view of the interface. Main residues shown as sticks; distances <3.4 Å shown as dashes. (C) SEC-MALLS on TPL202 (blue) and TPL202 tetramerization mutants in both tetramerization interfaces I (red) and II (yellow). dRI, differential refractive index; MW, molecular weight.

Fig. 3.

Hydrophobic groove 3 is implicated in the interaction with LxLxL EAR motifs. (A) Hydrophobic grooves (G1, G2, and G3) identified in TPL N terminus. (B and C) Y2H binding assay between TPL-202 (WT and mutants) with IAA12-DI domain (B) and WUSCHEL protein (C). n = 3 for all experiments. Error bars represent SD, ɸ for empty vector. (D) HTRF binding assays between His-TPL202 (WT and mutants) and MBP-IAA12 protein. HTRF-specific signal is reported. n = 3 for all experiments. Error bars represent SD. (E) Structure of AtTPL184 IAA27 peptide IAA27. a.u., arbitrary units.

Fig. 4.

Interaction of the EAR-motif with the G3 groove of AtTPL. (A) Position of the IAA27 peptide in AtTPL G3. (B) Overlay of IAA1 peptide bound to OsTPR2 on the complex between the IAA27 peptide and AtTPL184. (C) Analysis of AtTPL G3 residues involved in the interaction with IAA12 proteins by HTRF. (D) Analysis of AtTPL G3 residues involved in the interaction with IAA12 peptide by fluorescence anisotropy. a.u., arbitrary units.

Fig. 5.

Groove 3 and tetramerization are linked. (A) Characterization of the interaction between IAA12 EAR motif (FAM labeled) and TPL proteins impaired in tetramerization by fluorescence anisotropy assay. EC₅₀ shown below the graph (B) HTRF competition assays performed on an initial GST-IAA12/MBP-AtTPL202 complex by adding increasing amounts of His-tagged AtTPL202 wt and mutant proteins. IC₅₀ shown below the graph. a.u., arbitrary units.

Fig. 6.

Importance of TPL G3 groove and tetramerization in auxin signaling. In planta repression assay in Col-0 protoplasts containing the integrated DR5::VENUS reporter gene. A loss of repression activity is observable in TPL mutants impaired in their interaction with repressors, their tetramerization, or carrying the tpl-1 mutation. DR5 activity is reported (n = 200 protoplasts). Error bars correspond to the 95% confidence interval. Two variables with different letters are significantly different (corrected P values < 0.05; two-sided Kruskal-Wallis test). a.u., arbitrary units.