Determinants of affinity, specificity, and phase separation in a supramodule from Post-synaptic density protein 95

The post-synaptic density (PSD) is a phase-separated membraneless compartment of proteins including PSD-95 that undergoes morphological alteration in response to synaptic activity. Here, we investigated the interactome of a three-domain supramodule, PDZ3-SH3-GK (PSG) from PSD-95 using bioinformatics to identify potential binding partners, and biophysical methods to characterize the interaction with peptides from these proteins. PSG and the single PDZ3 domain bound similar peptides, but with different specificity. Furthermore, we found that the protein ADGRB1 formed liquid droplets with the PSG supramodule, extending the model for PSD formation. Moreover, certain mutations, introduced outside of the binding pocket in PDZ3, increased the affinity and specificity of the interaction and the size of liquid droplets. Other mutations within the ligand binding pocket lead to a new binding motif specificity. Our results show how the context in terms of supertertiary structure modulates affinity, specificity, and phase separation, and how these properties can evolve by point mutation.