Fig. 4: Contribution of the covalent mechanism to binding.
a Binding measurements with the original or saturated versions of 6R,R and 3R. 6R,R was tested in the competitive peptide displacement assay for the importance of the incubation time with three MAPKs: the measurements were taken right after setting up the binding reaction ( ~ 5 min; first row) or after 1 h (second row). Note that the competition binding profiles are unchanged. Lower panels show the results of similar experiments but using the saturated form of two cyclohexenone based compounds (6”R,R and 3”R). Note that these latter compounds could not compete well with reporter D-peptide binding even up to 1 mM concentration, suggesting that the C2 = C3 double bound is very important for binding as expected. (Kiapp values show the mean with parameter estimation error based on the least square method, n = 3; FB: fraction bound.). b Binding energy contribution of noncovalent and covalent mechanism in the binding of 8 to ERK2. The upper panels show the results of the peptide displacement assay using a weak binding reporter peptide (pepMK2) (FP: fluorescence polarization, FB: fraction bound; n = 3). Left panel: direct titration experiment with the ERK2 double mutant lacking both D-groove nucleophiles (C161A/H125L mutant); right panel: competitive titration with 8. Summary of the energy contribution of the noncovalent and covalent binding mechanisms is shown below. The values for ERK2 WT (Kiiapp ~4 μM) and for the single ERK2 C161A mutant (Kiapp ~160 μM) are taken from the experiment shown on Fig. 2b. The Kiapp value measured with the ERK2 C161A/H125L double mutant shows the noncovalent contribution (gray bar). Based on the scheme introduced on Fig. 1e, the Kchem´ value for ERK2 WT and 8 binding can be calculated. (FP: fluorescence polarization in arbitrary units; FB: fraction bound.). c 1H NMR spectra of the 3R-BME and 6 R,R–His-Test adducts. The left panel shows the structure of 3 R and of its thiol-adduct diastereomers (enol forms). Panels below show the 1H NMR spectra of the initial sample 3R (14.1 mM) and after mixing it with 1.4 molar equivalent BME and collected after 90 min. (The HC proton was in an overlapping position.) The panel on the right shows the structure of the 6R,R–His-Test covalent adduct (enol form) with the corresponding spectra for the His-Test sample alone (5 mM), the initial 6R,R sample (17.2 mM), and after mixing the latter with 2.6 molar equivalent His-Test (collected after 60 min). 1H NMR spectra were recorded in PBS (D2O; pH ~7.2) with 75% DMSO. The major stereoisomer of the imidazole adduct is R ( > 95 %). The 6.02 ppm protons from the reference compound was used as the internal standard (IS) important to be able to calculate Kchem(His) (n = 1; see Supplementary Note 1). d The structures of compounds analyzed by electronic circular dichroism (ECD) measurements with their Kchem(thiol) value determined based on BME titration experiments (colored parts of the molecules highlight important differences; a tert-butyl ester or amide in 3R vs 18R, a bridged skeleton in 6R,R, and anilide in 19’S). An example of this measurement for the 6R,R + BME reaction is shown on the panels right of the dashed vertical line (n = 1). These show the ECD spectrum of 6R,R (black) in PBS (pH ~7.4) compared with those produced by addition of different equivalents of BME (from 2 to 32 equivalents) and the inset figure shows the enlarged 275–410 nm wavelength range with 0–250 equivalents of BME; the concentration of 6R,R was 250 μM) and the right panel shows the change of the ECD signal of 6 R,R (∆ε) in the function of increasing concentration of BME monitored at 341 nm. Curve fitting produced the value of 409 ± 68 (M−1) for the equilibrium constant, corresponding to Kchem(thiol) value of ~2.5 mM for this compound (see Supplementary Note 2). Source data are provided as a Source Data file.
