Fig. 3. Binding site mapping and structural model of the phenothiazine-SPF45 UHM interaction.

a ¹H-¹⁵N HSQC experiments of 80 μM ¹⁵N-labeled SPF45 UHM domain free (black) and in the presence of 1.6-fold molar excess of 7,8-dihydroxyperphenazine (green) or 7,8-dimethoxyperphenazine (red). b Residues which show significant amide CSPs upon titration of 7,8-dimethoxyperphenazine are highlighted in red on the structure of the SPF45 UHM domain. c 2D ω₁-filtered NOEs between the SPF45 UHM domain and 7,8-dimethoxyperphenazine are shown. Red boxes indicate NOEs that were assigned and used in docking calculations. Chemical shift assignments of SPF45 protons and of the compound are shown on top and at the right-hand side of the spectrum, respectively. d Chemical structure of 7,8-dimethoxyperphenazine with atom numbering. e The lowest energy structure from the HADDOCK cluster is shown with the binding site residues. 7,8-dimethoxyperphenazine is colored in salmon.