Thousands of protein domains encoded in the human genome function by binding up to a million short linear motifs embedded in intrinsically disordered regions of other proteins. How affinity and specificity are encoded in these binding domains and the motifs themselves is not well understood. The evolvability of binding specificity - how rapidly and extensively it can change upon mutation - is also largely unexplored, as is the contribution of ‘fuzzy’ dynamic residues to affinity and specificity in protein-protein interactions. Here we produce the first global map of affinity and specificity encoding in a globular protein domain. Quantifying >200,000 energetic interactions between the domain and ligand allows us to identify 20 major energetically coupled pairs of sites. These are organised into six modules, with the vast majority of mutations in each module only reprogramming specificity for a single position in the ligand. Nine of the major energetic couplings encoding specificity are direct structural contacts and 11 have an allosteric mechanism of action. The dynamic tail of the ligand is more robust to mutation than the structured portion but contributes additively to binding affinity and communicates with structured residues to enable changes in specificity. Our results present how affinity and specificity are encoded in a globular protein domain interacting with a disordered peptide and a direct comparison of the encoding of affinity and specificity in structured and dynamic molecular recognition.
Overall design
Input and output samples of DNA amplicons from libraries of variants used to quantify fitness/protein binding scores of different variants of PSD95-PDZ3 and CRIPT proteins. Libraries were transformed into yeast and subjected to a protein complementation assay with or without a selective drug.
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