Counterintuitive Binding of Phosphorylated DEP Domain from Dishevelled Protein to Negatively Charged Membranes

To accomplish its role of signaling hub in all Wnt signaling pathways, Dishevelled (DVL) protein needs to dynamically relocalize to the inner leaflet of the cellular plasma membrane (PM). Combined experimental and computational evidence showed that the binding of DVL to the PM is mainly driven by the electrostatic attraction between a stretch of positively charged amino acids located on the C-terminal DEP domain of DVL and anionic phospholipid species, with a striking preference for phosphatidic acid (PA). Here, by means of computational simulations and QCM-D experiments, we demonstrate that four recently identified phosphorylation sites on DEP domain, alter the electrostatic potential of the membrane binding interface, but do not prevent the recruitment to anionic membranes. On the contrary, the phosphorylated residues are involved in hydrogen bond and ion-mediated interactions with the lipid headgroup of PA. Our results suggest that the effect of phosphorylation on protein-membrane association could be counterintuitive and sensitive to changes in the local environment including specific lipids, salts, and pH. SIGNIFICANCEPhosphorylation regulates the cellular activity and localization of many peripheral proteins by, among others, decreasing the affinity for negatively charged membranes. Here, we report how phosphorylation affects the membrane interaction of DEP domain from Dishevelled protein, the intracellular signaling hub in Wnt pathways. We found that despite the negative charge induced by phosphorylation, DEP domain was steadily adsorbed to the surface of negatively charged PA-rich membranes, due to a dense network of cation-mediated interactions and hydrogen bonds.