Fragment-Based Interrogation of the 14–3–3/TAZ Protein–Protein Interaction

The identification of chemical starting points for the development of molecular glues is challenging. Here, we employed fragment screening and identified an allosteric stabilizer of the complex between 14–3–3 and a TAZ-derived peptide. The fragment binds preferentially to the 14–3–3/TAZ peptide complex and shows moderate stabilization in differential scanning fluorimetry and microscale thermophoresis. The binding site of the fragment was predicted by molecular dynamics calculations to be distant from the 14–3–3/TAZ peptide interface, located between helices 8 and 9 of the 14–3–3 protein. This site was confirmed by nuclear magnetic resonance and X-ray protein crystallography, revealing the first example of an allosteric stabilizer for 14–3–3 protein–protein interactions.

TCF203 (Kd = 1272 ± 404 µM), and TCF347 (Kd = 166 ± 22 µM) binding to 14-3-3/TAZ complex (mean±SD; n=2).Analysis of TCF043 and TCF347 binding to 14-3-3/TAZ was carried out using fluorescence.(B) MST trace when IRlaser is off (blue line) and when the IR-laser is on (after blue line).Analysis was carried out using blue and red lines for fragments TCF199, TCF203 and TCF018.Fluorescence quenching is seen upon binding of TCF043 and TCF347.(C) Measurements of initial fluorescence at different fragments´ concentrations (concentration increases from left to right).Fragments TCF043 and TCF347 show fluorescence change upon binding.(D) Capillary scan after SD-test for fragments TCF043 and TCF347.Three highest and three lowest fragment concentrations were used for SD-test.For TCF043 a fourth capillary scan was excluded (grey).
Figure S3: Characterization of TCF199 binding to 14-3-3σΔC and 14-3-3σΔC/TAZ using NMR experiments.(A) WaterLOGSY experiments corresponding to TCF199 (2 mM, negative control in light blue), 14 -3-3σΔC in the apo form and 14-3-3σΔC complexed to TAZ (25 µM and 25µM/500µM, control experiments in dark blue and in green, respectively), apo-14-3-3σΔC in the presence of TCF199 (25µM/2 mM, in red) and 14-3-3σΔC in the presence of TAZ and of TCF199 (25µM/500µM/2 mM, in blue).The represented spectral region shows aromatic protons and signals detected from the TCF199 compound only (signals at 7.4 and 7.6 ppm not present in the 14 -3-3σΔC and 14-3-3σΔC/TAZ control experiments).Binding to 14-3-3σΔC and 14-3-3σΔC/TAZ was evidenced as a sign inversion of these signals from negative values in TCF199 alone control experiment (in light blue) to positive values for both apo-14-3-3σΔC in the presence of TCF199 (in red) and 14-3-3σΔC in the presence of TAZ and of TCF199 (in blue).(B) The increased intensity of these signals in the Water LOGSY spectrum of 14-3-3σΔC/TAZ/TCF199 compared to the corresponding signals in 14-3-3σΔC/TCF199 suggested an higher affinity of TCF199 for the complex.(C) 2D 1 H-15 N TROSY-HSQC spectra of 75 μM 15 N 2 H labeled 14-3-3σΔC in the presence of the TAZ peptide (in green) and in the absence (overlayed in dark blue).(D) Combined 1 H, 15 N chemical shift modifications between the spectra presented in (B), showing large chemical shift perturbations of multiple resonances following binding of the TAZ peptide.(E) 2D 1 H-15 N TROSY-HSQC spectra of 75 μM 15 N 2 H labeled 14-3-3σΔC in the presence of TCF199 (in red) and in the absence (overlayed in dark blue) (F) 2D 1 H-15 N TROSY-HSQC spectra of 75 μM 15 N 2 H labeled 14-3-3σΔC complexed to TAZ in the presence of TCF199 (in light blue) and in the absence (overlayed in green).(E-F) Resonance of H-15 N Thr217 that showed a small chemical shift value modification upon TCF199 binding is labelled.

Figure S4 :
Figure S4: MD simulations.A) Statistically significant binding site of TCF199 to the 13-3-3/TAZpS89 complex; TCF199 is shown as orange surface.B-C) Detail of the two possible orientations of TCF199 within the binding site identified by MD simulations.14-3-3 is shown as green cartoon, TAZpS89 is shown as magenta cartoon.In panels B-C, TCF199 is shown as cyan sticks, non-polar H atoms were omitted.

Figure S5 :
Figure S5: TCF199`s binding site analysis.Electron density before modeling (red circle; left-hand side) and after modeling (right-hand side).

Figure S6 :
Figure S6:Comparison of MD vs X-ray structure.Structural superimposition between the most representative frame extracted from MD trajectories (in green) and the crystallographic structure (in yellow) of the 14 -3-3/TAZpS89/TCF199 complex.

Table S1 -
D: Data collection and refinement statistics.Data collection statistics were calculated with Aimless and refinement statistics were extracted using the "table 1" tool of Phenix.Values in parenthesis correlate to high resolution shell.