Fig. 1
From: Modulating multi-functional ERK complexes by covalent targeting of a recruitment site in vivo
BI-78D3 labels C159 of ERK2. a Chemical structure of BI-78D3. b BI-78D3 inhibits ERK1/2 phosphorylation by constitutively active MKK1 (MKK1G7B) and inhibits the phosphorylation of Ets-1 by activated ERK1/2. Different concentrations of BI-78D3 were incubated with either unphosphorylated or activated ERK1/2 for 30 min before the addition of [γ- 32P] ATP and MKK1G7B or Ets-1, respectively (data are from three independent experiments for Ets-1 assays and two for MKK1G7B assays, and bars represent mean ± SD (standard deviation)). c The top panel shows an expansion of the region corresponding to C159 in 15N, 1H TROSY (600 MHz) spectra of inactive ERK2 (200 μM) in the presence of increasing amounts of BI-78D3. The bottom panel shows the change in the relative intensity of the C159 resonance in these spectra with increasing concentrations of BI-78D3. d Residues that show significant spectral perturbations calculated from 15N,1H TROSY (800 MHz) or 13C, 1H HMQC (800 MHz) spectra of ERK2 in the presence of approximately equimolar amounts of BI-78D3 for backbone amide (left panel) or Ile (δ1), Val and Leu methyls (right panel) resonances are indicated on the structure of ERK2. Residues for which the amide or methyl chemical shift perturbations exceed the corresponding average plus twice the standard deviation are colored dark green and cyan, respectively. Residues for which amide resonances that are broadened to below the noise are colored light green. C159 is shown in yellow (and labeled in blue) and the activation loop T183 and Y185 are shown in red stick representation. All of the spectral perturbations in both cases are centered in and around the DRS of ERK2. e Fluorescence anisotropy was employed to assess the ability of BI-78D3 to competitively displace a fluorescent D-site-containing peptide from the DRS of activated ERK2 and ERK2 C159S (represents one experiment out of two repetitions). f Specificity of BI-78D3 towards C159 and C164. ERK proteins (5 µM) were incubated with 100 µM BI-78D3 for 60 min. Free thiol was titrated using Ellman’s reagent (data are from three independent experiments, and bars represent mean ± SD)
