Fig. 3: Structural model of the ERK2-RSK2-ORF45 complex and PPI validation in cells. | Nature Communications

Fig. 3: Structural model of the ERK2-RSK2-ORF45 complex and PPI validation in cells.

From: A non-catalytic herpesviral protein reconfigures ERK-RSK signaling by targeting kinase docking systems in the host

Fig. 3

a Hydrogen Deuterium Exchange Mass Spectometry (HDX-MS) of the RSK2-ERK2 binary complex (in black) and RSK2-ERK2-ORF45 ternary complex (in red). Deuterium uptake of selected peptides are displayed onto the NTK from the NTK-ORF45 crystal structure or onto the CTK-ERK2 crystal structure (PDB: 4NIF). Data are represented as mean values ± SEM of three technical replicates. b Results of the SAXS analysis and the CORAL model of the ternary complex. The panel on top shows the experimental SAXS curve (black dots) with the simulated curve (in red) calculated based on the structural model shown on the panel below. Flexible N- and C-terminal elements (N or C) of RSK, the interdomain linker of RSK (colored in black), and the intervening region of the ORF45 peptide between the VF and FxFP motif regions are shown with thin ribbon and their Cα atoms are shown with spheres. The structural model was built using three binary crystallographic models (ppERK2-ORF45, NTK-ORF45, and CTK-ERK). c The ppERK2-RSK2-ORF45(16-76) ternary complex shown from two different orientations. The ORF45 is colored in magenta, the core FxFP and VF motifs are shown in yellow, and the intervening region between these two is shown with thin ribbon where Cα atoms are shown with spheres. The flexible interdomain linker between the NTK and CTK is indicated with a dashed line. N and C denotes N- and C-terminal residues of RSK where their Cα atoms are shown with spheres. The AMPPNP in the NTK nucleotide pocket is shown with black sticks and the active site residue (D193) is colored red. d Monitoring binary RSK2-ORF45 and ERK2-ORF45 binding in cells. The panel on the left shows the NanoBit luciferase signal for ORF45-RSK2 binary interaction. Middle panel: Normalized NanoBit signal for the ORF45-RSK2 interaction probes upon EGF treatment. Right panel: Normalized NanoBit signal for the ORF45-ERK2 interaction probes upon EGF treatment. The inset shows the same plot but without the curve for the ORF45(F66A)-ERK2 probe. WT (blue): full-length ORF45; F66A (red): F66 from the VF motif replaced to alanine; mFxFP (yellow): FxFP region is mutated to 4 alanines; F66A/mFxFP (green): both regions are mutated. The luminescence signal after EGF treatment was normalized to the signal from untreated cells (control). Error bars in light gray show SD based on three or six independent experiments. Data points show the mean ± SD. (N = 6, left panel; N = 3, middle and right panel; Paired t-test, two-sided; NS not significant, *p < 0.05, **p < 0.01, ***p < 0.001). Source data are provided as Source data file.

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