Research ArticleSTRUCTURAL BIOLOGY

Structural basis for recognition of diverse transcriptional repressors by the TOPLESS family of corepressors

See allHide authors and affiliations

Science Advances  24 Jul 2015:
Vol. 1, no. 6, e1500107
DOI: 10.1126/sciadv.1500107
  • Fig. 1 The N terminus of TPL/TPR proteins binds the NINJA EAR motif.

    (A) Schematic diagram of the domain structure of OsTPR2. In a search against the Protein Data Bank (PDB) database, we identified a seven-bladed β-propeller structure (PDB code: 3MXX) with a sequence identity of 26% to the first WD40 domain and 25% to the second WD40 domain of OsTPR2. Thus, the overall structure of the TPL/TPR C-terminal WD repeats can be modeled as two seven-bladed β-propeller domains. Q, P/G/T/Q-rich linker region. (B) Mammalian two-hybrid interaction between full length (FL), N-terminal domain (NTD; amino acids 1 to 209, 1 to 210, and 1 to 209), and CTDs {amino acids 211 [TPL(211–1133)] to 1133 [TPL(211–1133)]/210 to 1133 [TPR1(211–1133)]/210 to 1129 [TPR2(210 –1129)]} of rice TPL/TPR1/TPR2 proteins fused to VP16 transcriptional activation domain (VP16) and full-length NINJA fused to Gal4 DBD (Gal4-NINJA) (n = 3; error bars, SEM). UAS, upstream activating sequence; AD, activation domain; RLU, relative light units. (C) AlphaScreen luminescence proximity assay between the H6-NTD [same amino acids as in (A)] and H6-CTD (amino acids 323 to 1116/317 to 1113/316 to 1107) of rice TPL/TPR1/TPR2 proteins and biotinylated NINJA EAR motif peptide (b-NINJA) (n = 3; error bars, SD). (D) AlphaScreen interaction between the OsTPR2 N-terminal domain and biotinylated wild-type (WT) and mutant (3A) NINJA peptide or biotinylated MBP-tagged full-length NINJA protein. (E) AlphaScreen interaction between H6-tagged TPL/TPR N-terminal domains and b-NINJA.

  • Fig. 2 The OsTPR2 TPD forms a novel tetrameric fold.

    (A) The OsTPR2 TPD forms a tetramer. Left: Cartoon diagram of the tetrameric structure of OsTPR2 TPD with the LisH and CTLH motifs colored in blue and brown, respectively. The Zn2+ ions are shown as gray spheres. Right: Surface structure of the tetramer. (B) Structure of an OsTPR2 TPD monomer in rainbow color scheme from N terminus (N; blue) to C terminus (C; red). The secondary structure diagram of the OsTPR2 TPD fold is shown on the right.

  • Fig. 3 The OsTPR2 TPD tetramer has two distinct dimerization interfaces.

    (A) View of the TPR2 TPD tetramer with boxed interfaces 1 and 2. (B) Overview of the dimerization domain 1. Dimerization interface 1 is formed by the N-terminal four-helix bundle of the LisH domain and the C-terminal helix α9. The two helices from each monomer forming the LisH domain are shown in blue. (C) A close-up view of the interface with key interacting residues shown as stick model and interactions as dashed lines. (D) Interface 2 with key interacting residues shown as stick model and interactions as dashed lines. (E) The interaction between α6 K102 from one monomer and α7 T120 from the other monomer induces a kink in α6 for antiparallel α6-α7 packing. This fold represents a novel dimerization motif. A structure homology search revealed an artificial, rationally designed homodimerization motif (PDB: 3V1F) as closest structure homolog in the PDB.

  • Fig. 4 Four EAR motif peptides bind the hydrophobic surface grooves of one OsTPR2 TPD tetramer.

    (A) Tetrameric structure of OsTPR2 TPD complexed with NINJA EAR motif peptides. The peptide binding sites are indicated by black dashed circles with peptides shown in stick representations. (B and C) Close-up views of a NINJA EAR motif (stick presentation) bound to the OsTPR2 TPD peptide-binding groove shown as cartoon presentation (B) or as charge potential surface (C) (blue, positive charge potential; red, negative charge potential). A color code bar (bottom) shows the electrostatic scale from –5 to +5 eV. (D) Structure overlays of the EAR motifs from NINJA, IAA1, and IAA10 in the OsTPR2 TPD surface pocket.

  • Fig. 5 Structure of OsTPR2 TPD in complex with NINJA EAR peptide.

    (A) Close-up view with charge interaction, hydrogen bond, and hydrophobic interactions shown as black dashed lines. The NINJA peptide sequence is shown above the graph. The residue carbon atoms in the OsTPR2 TPD hydrophobic binding cleft are labeled in cyan, whereas the residue carbon atoms of the EAR motif are labeled in green for the three conserved leucine residues and gray for the rest of residues. (B) 2FoFc electron density map contoured at 1σ of residues at the OsTPR2 TPD–NINJA EAR motif interaction interface.

  • Fig. 6 Mutations of OsTPR2 TPD–NINJA EAR motif interface residues abolish interaction between full-length OsTPR2 and NINJA in a mammalian two-hybrid assay.

    (A) Stick presentation of NINJA and OsTPR2 interaction residues. The binding groove is shown as transparent surface. (B) Mutations of key residues of the OsTPR2 TPD hydrophobic cleft disrupt the interaction between full-length OsTPR2 and NINJA. (C) Effect of mutations of NINJA EAR motif residues on the OsTPR2-NINJA interaction.

  • Fig. 7 EAR motifs differentially interact with OsTPR2 TPD.

    (A) Relative OsTPR2 affinities of different EAR motifs. Affinities were determined by AlphaScreen competition curves (fig. S11) under conditions where the IC50 values approximate the dissociation constants (Kd) of the interactions. (B) Mutations of IAA10 EAR motif residues reduce interaction between EAR peptides and OsTPR2 TPD. Relative affinities were determined by AlphaScreen competition curves (fig. S12) under conditions where the IC50 values approximate the Kds of the interactions.

  • Fig. 8 Repressor oligomerization can greatly increase TPD affinity.

    (A) Cartoon presentation of the interaction between tetrameric OsTPR2 TPD and either a monomeric 12–amino acid version of NINJA EAR (NINJA: DNGLELSLGLSC) or tetrameric TraM-NINJA EAR. Weak EAR-TPD interactions (black double-sided arrows) are greatly stabilized by simultaneous TraM EAR–TraM EAR (red arrows) interactions, resulting in a much more than additive increase in OsTPD binding affinity. (B) TraM-NINJA EAR forms a tetramer in solution. SEC elution profile with elution volumes of size standards indicated. Each monomer has a size of 10.9 kD. mAU, milliabsorbance units. (C) Homologous AlphaScreen competition curves of the interactions between H6-OsTPR2 TPD and either biotin-TraM-NINJA EAR (blue squares) or biotin-NINJA EAR peptide (red circles).

  • Fig. 9 Model of the recruitment of TPL/TPR corepressors by EAR motifs.

    The EAR motifs shown are from adaptor proteins (NINJA, jasmonate signaling), repressors (IAA proteins, auxin signaling), and TFs [BZR1 (brassinazole resistant 1), brassinosteroid signaling]. The OsTPR2 TPD is shown as a space-filling structure based on this study, whereas the WD40 seven-bladed β-propeller domains are depicted as ribbon models based on the structure of the yeast Tup1 C terminus (PDB code: 1ERJ). TFs are shown in green, repressors in red, and the adaptor protein NINJA in blue. This model does not imply that EAR repressors from different signaling pathways can bind together on a single TPL tetramer but rather that different types of EAR-containing transcriptional proteins share the same TPL/TPR binding mode and may compete for TPL/TPR binding in vivo. HMT, histone methyltransferase; HDAC, histone deacetylase.

  • Table 1 X-ray diffraction data and refinement statistics for TPR2 TPD structures.
    SeMet–TPR2 TPDApoTPR2 TPDTPR2 TPD + NINJA complexTPR2 TPD + IAA10 complexTPR2 TPD + IAA1 complex
    Data collection
    Space groupP212121P42212P21P3121P21
    Cell dimensions
      a, b, c (Å)70.2, 111.9, 145.059.0, 59.0, 171.781.7, 65.0, 107.7162.7, 162.7, 157.358.4, 129.1, 79.1
      α, β, γ (°)90, 90, 9090, 90, 9090, 105.4, 9090, 90, 12090, 110, 90
    Wavelength0.9787 (peak)1.278*1.0781.0780.9787
    Resolution
      All (Å)50–2.550–3.2550–3.150–3.150–2.7
      Last shell (Å)2.64–2.53.43–3.253.31–3.13.27–3.12.85–2.7
    Rsym or Rmerge0.109 (1.0)0.081 (1.26)0.172 (0.566)0.074 (0.93)0.072 (0.678)
    II20.4 (3.2)27.5 (3.5)5.9 (2.2)18.3 (2.0)15.0 (2.0)
    Completeness (%)100 (100)100 (100)99.9 (99.9)99.9 (99.9)99.9 (99.9)
    Redundancy14.8 (15.0)26.3 (28.2)4.1 (4.2)7.4 (7.6)5.2 (4.3)
    Refinement
    Resolution (Å)50–2.550–3.2550–3.150–3.150–2.7
    No. of reflections76,2129,10320,02043,93930,324
    Rwork/Rfree0.199/0.2420.212/0.2690.232/0.2860.196/0.2290.22/0.252
    No. of molecules per asymmetric unit41464
    No. of atoms
      Protein6,8041,7036,69210,2916,620
      Ligand/peptide01188344241
      Water3784342389
    B-factors
      Protein60.0119.057.4101.269.1
      Ligand/peptideN.A.105.568.2127.879.8
      Water54.154.141.588.363.3
    RMSDs
      Bond lengths (Å)0.0120.0080.0080.0080.009
      Bond angles (°)1.461.281.411.441.37
    Ramachandran
      Favored (%)98.198.098.998.499.0
      Outliers (%)0.00.00.00.240.37

    *Native crystal data were collected at this wavelength to measure Zn anomalous signal.

    †Values in parentheses are for the highest-resolution shell.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/6/e1500107/DC1

      Fig. S1. Sequence alignment of the N-terminal region of different transcriptional repressors, TFs, and adapters that interact with TPL/TPR.

      Fig. S2. Charge distribution surface views of the complex between OsTPR2 TPD and the NINJA EAR motif.

      Fig. S3. Biochemical and stuctural characterization of the TPR2 TPD tetramer.

      Fig. S4. The rOsTPR2 protein contains a zinc finger structure.

      Fig. S5. Structural overlay of OsTPR2 TPD apo and NINJA peptide–bound structures.

      Fig. S6. Sequence alignment of the TPD of TPL and TPR proteins from rice (Os) and Arabidopsis (At).

      Fig. S7. Structure of OsTPR2 TPD in complex with IAA10 EAR peptide.

      Fig. S8. Structure of OsTPR2 TPD in complex with IAA1 EAR peptide.

      Fig. S9. Structural comparison of NINJA, IAA10, and IAA1 peptide–bound OsTPR2 TPD structures.

      Fig. S10. OsTPR2 TPD N176H forms higher-order oligomers.

      Fig. S11. Relative TPD affinities of different EAR motifs.

      Fig. S12. Effects of EAR point mutations on TPD binding.

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. Sequence alignment of the N-terminal region of different transcriptional repressors, TFs, and adapters that interact with TPL/TPR.
      • Fig. S2. Charge distribution surface views of the complex between OsTPR2 TPD and the NINJA EAR motif.
      • Fig. S3. Biochemical and structural characterization of the TPR2 TPD tetramer.
      • Fig. S4. The rOsTPR2 protein contains a zinc finger structure.
      • Fig. S5. Structural overlay of OsTPR2 TPD apo and NINJA peptide–bound structures.
      • Fig. S6. Sequence alignment of the TPD of TPL and TPR proteins from rice (Os) and Arabidopsis (At).
      • Fig. S7. Structure of OsTPR2 TPD in complex with IAA10 EAR peptide.
      • Fig. S8. Structure of OsTPR2 TPD in complex with IAA1 EAR peptide.
      • Fig. S9. Structural comparison of NINJA, IAA10, and IAA1 peptide–bound OsTPR2 TPD structures.
      • Fig. S10. OsTPR2 TPD N176H forms higher-order oligomers.
      • Fig. S11. Relative TPD affinities of different EAR motifs.
      • Fig. S12. Effects of EAR point mutations on TPD binding.

      Download PDF

      Files in this Data Supplement:

    Stay Connected to Science Advances

    Navigate This Article