Viral PDZ target sequences are not inherently more promiscuous than those derived from endogenous target proteins.

PDZ domains are one of the largest families of short linear motif (or peptide) binding domains in the human proteome. These scaffolding domains are important in signaling and trafficking pathways, such as in the formation of tight junctions or in the postsynaptic density of neurons. PDZ domains are also targeted by several pathogenic viral proteins, including human papillomavirus (HPV), influenza, hepatitis, rabies, coronaviruses, among others. Previously, we investigated the specificity determinants of modulator, or non-motif, residues for two PDZ binding motifs (or PBMs) from the cystic fibrosis transmembrane conductance regulator (CFTR) and HPV16 E6 proteins to better understand differences in relative promiscuity between these similar sequences. To test whether viral PDZ-binding motifs are inherently more promiscuous than endogenous targets, we measured binding affinities for 7 endogenous and 7 viral PBMs across 8 well-studied Class I PDZ domains. Fluorescence anisotropy assays revealed no significant difference in relative binding affinities between viral and endogenous PBMs, indicating that viral versus endogenous origin alone does not explain PBM promiscuity. Our results were consistent with available data from high throughput holdup assays. Taken together, our data support previously reported sequence hotspots, whereby certain PBMs are recognized by large numbers of PDZ domains, likely due to a combination of widely favorable modulator residues.