Research ArticleBiochemistry

Substrate binding allosterically relieves autoinhibition of the pseudokinase TRIB1

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Science Signaling  25 Sep 2018:
Vol. 11, Issue 549, eaau0597
DOI: 10.1126/scisignal.aau0597
  • Fig. 1 The TRIB1-C/EBPα degron complex.

    (A) Schematic illustrating degradation of C/EBPα by TRIB1-COP1. (B) Crystal structure of the TRIB1-C/EBPα degron complex, with TRIB1 shown predominantly in orange with a green activation loop and C/EBPα in blue. (C) Comparison of the C/EBPα binding mode (blue) with that of the prototypic substrate-like PKI (turquoise) in complex with PKA [gray surface; Protein Data Bank (PDB) 1ATP]. To generate the overlay, the TRIB1-C/EBPα complex structure was superimposed on the basis of the pseudokinase and kinase domains of TRIB1 and PKA, respectively. (D) Glutathione S-transferase (GST) pulldown of His6MBP-C/EBPα(53–75) by wild-type (WT) GST-TRIB1(84–372) and indicated mutants, separated by SDS–polyacrylamide gel electrophoresis (PAGE) and visualized by Coomassie blue staining (top) or anti-His6 immunoblotting (bottom). A nonspecific band that copurifies with GST-TRIB1 is indicated with an asterisk. (E) Structural representation of mutants that disrupt C/EBPα binding. TRIB1 is shown as an orange surface and C/EBPα in blue, with TRIB1 mutants that disrupt binding shown in red.

  • Fig. 2 TRIB1 binds various C/EBPs and can be antagonized by phosphorylation.

    (A) Alignment of the TRIB degron from various human C/EBP proteins, and the Drosophila C/EBP ortholog, Slbo. (B) ITC of indicated MBP-fused C/EBP degrons injected into TRIB1(84–372). Data points represent mean of duplicate titrations, following buffer titration subtraction. (C) Detailed view showing the integral role of Ser65 and Tyr67 in C/EBPα degron binding. Phosphorylation has been reported on the equivalent residues in C/EBPβ (Ser77 and Tyr69). (D) Fluorescence polarization displacement assay of FITC-C/EBPα degron from TRIB1 by unmodified, Ser77-phosphorylated, or Tyr79-phosphorylated C/EBPβ peptide. Data are means ± SEM of three independent replicates from one purified TRIB1 stock.

  • Fig. 3 TRIB1 conformational changes upon substrate binding.

    (A to D) Comparison of structures of substrate-free (top; PDB ID 5CEM) and C/EBPα-bound TRIB1 (bottom; this work). (A) Overview from the substrate-binding side of the molecule. (B) SLE motif and surrounding residues within the active site from the same orientation as (A). (C) Simplified view showing the relative organization of the αC helix, Tyr134, and Leu226 from the opposite orientation to (A) and (B). (D) Position of the αC helix and residues that contact the C-terminal tail of TRIB1 in substrate-free TRIB1; orientation as described in (C). Movement of selected features upon C/EBPα binding are indicated with arrows (top), and residues are selectively depicted as sticks for clarity.

  • Fig. 4 Allosteric release of the COP1-binding motif by substrate binding.

    (A and B) Structure of the C-terminal COP1-binding motif bound to (A) the pseudokinase domain of TRIB1 (from PDB ID 5CEM) or (B) the WD40 domain of COP1 (from PDB ID 5IGO). (C) Fluorescence polarization (Fp) displacement assay of FITC-TRIB1(349–367) from the WD40 domain of COP1 by either TRIB1(84–372) alone at varying concentrations (above arrow in the diagram; black in the graph) or TRIB1(84–372) in the presence of 10 μM C/EBPα degron peptide (below arrow in the diagram; yellow in the graph). Data are means ± SEM of three technical replicates using two independently prepared stocks of COP1. (D) Summary of MD simulations of SLE-out TRIB1 or SLE-in TRIB1 with and without substrate. The number of hydrogen bonds between the C-terminal tail and β4 strand is plotted over the time course of the simulation above, and an indicative state during the latter part of the simulation is shown below. Animated trajectories are shown in movie S2.

  • Fig. 5 Comparative analysis of MD simulations of TRIB1 pseudokinase domain with and without C/EBPα peptide.

    (A) The KL divergence of the torsion angle of each residue is shown. Motifs and residues that display differential distribution between the C/EBPα-unbound simulation and C/EBPα-bound simulation are labeled. Left: KL divergence of TRIB1 simulations without the C-terminal tail. Right: KL divergence of TRIB1 simulations with the C-terminal tail. (B) Representative snapshots from TRIB1+C-tail:C/EBPα simulation. SLE+1 aspartate (Asp228) mediates charge interaction with Lys210 from catalytic loop, Arg102 from glycine-rich loop, and Lys130 from αC helix.

  • Fig. 6 TRIB1 can potentially bind small-molecule ligands but not ATP.

    (A and B) Representation of the available binding cavity of TRIB1 (gray) in (A) the SLE-out conformation from PDB ID 5CEM and (B) the SLE-in conformation when bound to C/EBPα. (C) DSF melting analysis of TRIB1 or TRIB1 + C/EBPα degron peptide in the presence or absence of ATP. Points are the mean of independent triplicates from one purified TRIB1 stock, with error bars representing SEM. (D) Summary of DSF analyses of TRIB1(84–372) against the Published Kinase Inhibitor Set (PKIS) library. Tm is expressed as the difference between each compound and the mean of three dimethyl sulfoxide (DMSO)–only controls. The four compounds shown in more detail in (E) are indicated. (E) Individual DSF melting curves of four selected compounds (left), with additional titrations for each that include C/EBPα degron peptide (right).

  • Table 1 Summary of crystallographic data and refinement.

    Rmerge = ∑hkli |Ii − <I> |/−∑hkl ∑Ii, where Ii is the intensity of the ith observation, <I> is the mean intensity of the reflection, and the summations extend over all unique reflections (hkl) and all equivalents (i), respectively. Rpim is a measure of the quality of the data after averaging the multiple measurements and Rpim = ∑hkl [n/(n − 1)]1/2i |Ii(hkl) – <I(hkl)>|/∑hkli Ii(hkl), where n is the multiplicity (other variables as defined for Rmerge).

    TRIB1-C/EBPα
    BeamlineAS-MX2
    Wavelength (Å)0.9537
    Resolution (outer shell) (Å)48.7–2.8 (2.95–2.8)
    Space groupP6122
    Unit cell parametersa = 98.8 Å
    b = 98.8 Å
    c = 332.7 Å
    α = 90°
    β = 90 °
    γ = 120°
    Rmerge (outer shell)0.077 (1.992)
    Rpim (outer shell)0.035 (0.879)
    Mean II (outer shell)19.8 (1.5)
    Completeness (outer shell)99.8 (99.1)
    Multiplicity (outer shell)10.4 (10.7)
    Total no. of reflections258,141 (37,340)
    No. of unique reflections24,777 (3,491)
    Mean (I) half-set correlation CC(1/2) (outer shell)1.000 (0.609)
    Wilson B factor (Å2)82.1
    Refinement statistics
      Rcryst0.220
      Rfree0.276
    Average B factor overall (Å2)113
    Ramachandran plot statistics (%)
      Favored regions93.1%
      Allowed regions6.5%
      Outliers0.4%
      PDB entry6dc0

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/549/eaau0597/DC1

    Fig. S1. Construct design for crystallization.

    Fig. S2. C/EBPα degron electron density.

    Fig. S3. Dissociation constant of TRIB1/2-binding C/EBPα.

    Fig. S4. Differing C/EBPα-binding potential between TRIB1 and TRIB3.

    Fig. S5. Stable regions of C/EBPα degron in complex with TRIB1.

    Fig. S6. Displacement of the TRIB1 C-terminal tail from COP1.

    Fig. S7. Destabilization of TRIB1 by Y134C mutation.

    Fig. S8. PKI screening of TRIB1.

    Table S1. DSF data for individual PKIS compounds.

    Movie S1. Morph between substrate-free (SLE-out) and C/EBPα-bound (SLE-in) structures of TRIB1.

    Movie S2. Simulations of the RIB1–C-terminal tail.

  • The PDF file includes:

    • Fig. S1. Construct design for crystallization.
    • Fig. S2. C/EBPα degron electron density.
    • Fig. S3. Dissociation constant of TRIB1/2-binding C/EBPα.
    • Fig. S4. Differing C/EBPα-binding potential between TRIB1 and TRIB3.
    • Fig. S5. Stable regions of C/EBPα degron in complex with TRIB1.
    • Fig. S6. Displacement of the TRIB1 C-terminal tail from COP1.
    • Fig. S7. Destabilization of TRIB1 by Y134C mutation.
    • Fig. S8. PKI screening of TRIB1.
    • Legend for table S1
    • Legends for movies S1 and S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). DSF data for individual PKIS compounds.
    • Movie S1 (.mp4 format). Morph between substrate-free (SLE-out) and C/EBPα-bound (SLE-in) structures of TRIB1.
    • Movie S2 (.mp4 format). Simulations of the RIB1–C-terminal tail.

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