Research ArticleBiochemistry

Structure of the C-terminal guanine nucleotide exchange factor module of Trio in an autoinhibited conformation reveals its oncogenic potential

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Science Signaling  19 Feb 2019:
Vol. 12, Issue 569, eaav2449
DOI: 10.1126/scisignal.aav2449
  • Fig. 1 Structural overview of the DH-PH interface found in TrioC in comparison with related DH/PH modules.

    (A) Overall domain orientation and structural layout of the autoinhibited TrioC DH/PH module. The DH domain is shown in green and is composed of α-helical segments α1 to α6. The PH domain is shown in cyan and is composed of αN and αC helices and β-strands 1 to 7. αN is colored orange, and the β3-β4 loop is colored magenta. (B and C) For comparison, the structures for active p63RhoGEF (PDB entry 2RGN) (B) and Dbs (PDB entry 1RJ2) (C) were aligned to TrioC based on their DH domains. Insets highlight the side chains of key residues in the DH-PH interface as sticks, except for Gly2149 in TrioC and Gly340 in p63RhoGEF, whose Cα atoms are shown as black spheres. In (B), Gαq·GDP·AlF4 is shown as a gray surface representation, and RhoA is omitted for clarity. In (B) and (C), the β3-β4 loops are partially disordered.

  • Fig. 2 Conformational changes that lead to occlusion of the RhoA binding site in TrioC.

    (A) TrioC is shown aligned with the activated structure of p63RhoGEF using their DH domains. TrioC exhibits a 60° rotation of its PH domain (cyan) relative to that of activated p63RhoGEF (dark gray). Inset: The observed conformation of TrioC forms steric overlaps with the RhoA binding site in two key regions, α6-αN and β3-β4, demarcated by asterisks. (B) The α6-αN junction also bends toward the DH domain by 30° in autoinhibited TrioC relative to related DH/PH structures of known structure. The hinge regions of autoinhibited TrioC, Gαq-p63RhoGEF-RhoA (PDB entry 2RGN), Dbs (PDB entry 1RJ2), and Dbs-RhoA (PDB entry 1LB1) are overlaid. Key TrioC residues are shown as sticks or spheres, as in Fig. 1. The analogous residues in p63RhoGEF are also shown.

  • Fig. 3 HDX-MS solution dynamics of the activated R2150W variant suggests physical separation of the DH and PH domains.

    Difference in HDX rates (TrioC R2150W-Trio WT) was used to color the Cα atoms in the TrioC crystal structure. Scale bar indicates the color that corresponds to a given rate of exchange, with red indicating more exchange in the variant compared to WT. The side chains of Glu2069 and Arg2150 are shown as sticks and the Cα atom of Gly2149 as a sphere. Data are the mean of n = 2 experiments (using protein from independent purifications) using matched peptides from HDXaminer and averaged over five time points. Plots of HDX on the primary structures of TrioC WT and R2150W are shown in fig. S3.

  • Fig. 4 Trio mutants found in patient tumors activate RhoA in HEK293 cells.

    (A) Quantification of n = 3 (n = 4 for G2149W) independent biological Rhotekin pull-down experiments for control condition (pEGFP-C1), Trio WT, and Trio variants with error shown as 95% CIs. Blots for RhoA·GTP were normalized using total RhoA content in cell lysate. Statistical comparisons to Trio WT were made using a one-way ANOVA test with a post hoc Dunnett’s test for multiple comparisons. *P < 0.05 and **P < 0.01. (B) Representative Western blot images from one of the three experiments. The top two blots show RhoA·GTP and total RhoA content, respectively. The third blot shows Trio variant expression, blotting for the eGFP fusion partner, with β-actin used as a loading control. Quantitative comparison of Trio variant expression over three independent biological experiments is shown in fig. S4. A minor truncation product of full-length Trio was present.

  • Fig. 5 Model for TrioC activation.

    TrioC exists in a conformational equilibrium between inactive and active states that can be biased toward the active state by either active Gαq or mutations in the DH-PH interface. The thicker half-arrows represent the favored direction in the equilibria. The DH domain is represented by green ovals with its RhoA binding site highlighted in yellow. α6 is shown as green rectangles that form a continuous helix with αN, represented as blue rectangles. The PH domain is represented as blue circles, with its C-terminal αC helix as black helices. Arg2150 is shown as a ball-and-stick model, and the β3-β4 loop is shown as a cartoon loop. Gαq is shown as gold shapes with its effector binding region in light yellow. Disorder is indicated with dashed lines and a blurring of Arg2150. The autoinhibited conformation in the top left quadrant is represented by PDB 6D8Z, and the maximum activity state is represented by PDB 2RGN, where Gαq binds to both the PH and DH domains and constrains them in a more open configuration that features a bent α6-αN helix.

  • Table 1 Crystallographic data collection and refinement statistics.

    X-ray crystallographic data for TrioC. Parentheses indicate values for highest resolution shell. ND, not determined; N/A, not available.

    TrioCΔC (human Trio
    1960–2275)
    Data collection statistics
    Synchrotron sourceLS-CAT beamline 21-ID-G,
    advanced photon source
    Wavelength (Å)0.97856
    Resolution range (Å)50–2.65 (2.70–2.65)
    Space groupP212121
    Unit cell (a, b, c) (Å)59.2, 85.8, 182.4
    Unique reflections30511 (1371)
    Multiplicity5.0 (3.8)
    Completeness (%)98.7 (90.1)
    Mean I/σI18.9 (1.2)
    CC1/2ND (0.699)
    Rsym0.085 (0.746)
    Refinement statistics
    Resolution limits (Å)15–2.65 (2.70–2.65)
    Number of test reflections28787 (1862)
    Rwork0.23 (0.34)
    Rfree0.27 (0.37)
    Number of nonhydrogen atoms7525
      Macromolecule7499
      Ligand0
      Water26
    Protein residues907
    RMS bonds (Å)0.008
    RMS angles (°)1.2
    Ramachandran favored (%)97.8
    Ramachandran outliers (%)0
    Clashscore calculated from
    MolProbity
    1.33
    Average B-factor76.0
      Macromolecule76.0
      LigandN/A
      Water48.0
  • Table 2 Biochemical analysis of the TrioC module.

    DSF and GEF activation data for TrioC variants. ΔTm = Tm(variant) − Tm(WT). Fold GEF activation = average kobs(variant)/kobs(WT). Fold activation by Gαq·GDP·AlF4 = average kobs(variant + Gαq·GDP·AlF4)/kobs(variant + Gαq·GDP). Each variant was profiled in n = 3 experiments, each performed at least in technical duplicate. A one-way analysis of variance (ANOVA) with post Dunnett’s test was used to test for significance for ΔTm, fold GEF activation, and Gαq·GDP·AlF4 activation for each variant in comparison to WT. See Materials and Methods for further explanation of methods and statistical analysis used for this table.

    VariantTm (°C)95% CI for ∆TmGEF activation
    (fold/WT)
    95% CI for GEF
    activation
    Fold activation by
    q·GDP·AlF4
    95% CI for fold
    activation by
    q·GDP·AlF4
    WT0.0[−0.5,0.5]†1.0[0.9, 1.1]3.1[0.4, 5.9]
    E2069A−6.0***[−7.3, −4.7]0.7[0.1, 1.3]NDND
    M2146A−3***[−3.6, −2.4]0.8[−0.5, 2.2]NDND
    S2208A−3.2***[−4.8, −1.7]1.4[0.1, 2.7]NDND
    R2150A0.9*[−0.2, 1.9]0.9[0.03, 1.8]NDND
    F2207A1.1[−0.6, 2.8]0.7[−0.1, 1.6]NDND
    ∆2204–2208−0.4[−1.1, 0.2]1.5[0.2, 2.8]NDND
    ∆2203–2209−0.5[−0.9, −0.1]1.0[−0.5, 2.7]NDND
    G2149I−6.3***[−6.7, −5.9]1.9[−0.7, 4.6]NDND
    E2069R/R2150E−7.9***[−14, −2.2]3.2**[1.6, 4.7]NDND
    R2150E0.9[0.7, 1.1]1.0[−0.4, 2.4]NDND
    2153ΔNDND3.0*[0.7, 5.3]NDND
    2152ΔNDND11***[4.5, 18]NDND
    2147ΔNDND14***[4.8, 22]NDND
    2143ΔNDND0.2[−0.2, 0.6]NDND
    G2149W−3.6***[−3.9, −3.3]4.3***[2.4, 6.4]1.5*[0.7, 2.3]
    R2150Q−1.6***[−3.3, 0.2]4.5***[2.1, 6.9]2.2[1.2, 3.1]
    R2150W−3.1***[−5.1, −1.2]9.3***[−0.4, 18]0.9**[0.7, 1.0]

    †Data were collected using two experimental setups; the larger 95% confidence interval (CI) was chosen here.

    *P < 0.05.

    **P < 0.01.

    ***P < 0.005.

    Supplementary Materials

    • This PDF file includes:

      • Fig. S1. Lattice contacts in the TrioC crystal structure.
      • Fig. S2. Sequence alignment of TrioC and related GEF modules.
      • Fig. S3. HDX-MS data for TrioC R2150W.
      • Fig. S4. Quantification of Trio variant expression in HEK293 cells.

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