Exploration of Peptide Recognition using Directed Evolution of the PDZ Domain Fold

The PDZ domain family is one of the most abundant peptide recognition modules in metazoan proteomes. Characterization of natural PDZ domains has provided insight into the structural basis and diversity of peptide recognition by this fold. In order to test the limits of the current model, I evolved synthetic PDZ domains. Based on the Erbin PDZ domain and selected for binding to pep- tides with different position-2 residues, synthetic variants were characterized using high throughput peptide profiling. This approach generated insight into subclass specificities in the most common natural specificity classes (I [ST]-2 and II Φ-2), demonstrated an alternative basis for a rare specific- ity (Class III D-2) and predicted that some natural domains may exhibit an easily-evolved novel specificity (Class IV R-2). These results also emphasize that some non-contact residues may have a disproportionate effect on position-2 specificity, contrary to the predictions of the original model.MAS