Fig. 6
PDZ phosphorylation regulates the interaction with the DVL C-terminal peptide. a Structure of DVL3 C-terminal peptide (aa 702–716; green) bound to the PDZ-binding pocket (PDZ domain from DVL3, aa 245–338; in silico ɑ-helices in dark and β-strands in light gray) observed in the in silico simulations. S268, S311, and C-terminus residue E710 participating in hydrogen bonds (dotted line) are highlighted with a stick model. b The matrix of mean interaction energy between each residue of PDZ domain aa 245–338 (x-axis) and DVL C-terminal peptide aa 709–716 (y-axis). Strength of the attraction of PDZ wt and C-terminus is depicted in white-black gradient. c–e The differences in mean interaction energies shown as a difference from wild-type PDZ wt: c PDZ (S268E/S311E), d PDZ (phospho-S268/phospho-S311), and e PDZ (S268A/S311A) between PDZ mutants. The change in the interactions are depicted in green (stronger) or red (weaker). Interactions of protein/peptide end caps are also displayed in the matrix. f–i NMR titrations of the DVL2 PDZ wild type and PDZ phosphomimicking variant (S286E/S329E; corresponding to S268 and S311 in DVL3) with DVL C-terminal peptide. f Overlay of 1H, 15N HSQC spectra for each titration point. Arrows indicate selected residues that exhibit fast exchange properties on the NMR timescale and gray boxes selected residues that exhibit intermediate-to-fast exchange properties on the NMR timescale. g Mapping of chemical shift perturbations on DVL-peptide (in green) structure from PDB database (PDB ID: 3CCO). Fast exchange residues colored using a gradient from white to red according to chemical shift perturbation and intermediate exchange residues that go beyond detection at the end of the titration colored in gray. S286, or E286 substitution, of DVL2 are highlighted by black arrow. h Binding isotherms for three residues that experience fast exchange during NMR titration. The apparent KD values represent the mean with the standard deviation for the three cross-peaks analyzed. i Experimental line shapes during the titration for two selected residues that experience intermediate-to-fast exchange. j SPR sensograms of DVL C-terminal peptide binding to wild type and S286E/S329E PDZ from DVL2 and the corresponding binding isotherms fitted to a one-site binding model. RU stands for response units, AU for arbitrary units
