Design of a Phosphorylatable PDZ Domain with Peptide-Specific Affinity Changes

Phosphorylation is one of the most common posttranslational modifications controlling cellular protein activity. Here, we describe a combined computational and experimental strategy to design new phosphorylation sites into globular proteins to regulate their functions. We target a peptide recognition protein, the Erbin PDZ domain, to be phosphorylated by cAMP-dependent protein kinase. Comparing the five successful designs to the unsuccessful cases, we find a trade-off between protein stability and the ability to be modified by phosphorylation. In two designs, Erbin's peptide binding function is modified by phosphorylation, where the presence of the phosphate group destabilizes peptide binding. One of these showed an additional switch in specificity by introducing favorable interactions between a designed arginine in the peptide and phosphoserine on the PDZ domain. Because of the diversity of PDZ domains, this opens avenues for the design of related phosphoswitchable domains to create a repertoire of regulatable interaction parts for synthetic biology. [Display omitted] ▸ Successful design of phosphorylation sites into a globular protein ▸ Redesigned PDZ domain phosphorylation is associated with local destabilization ▸ Phosphorylation switches peptide binding function ▸ Phosphorylation switches peptide recognition specificity Phosphorylation is one of the most common posttranslational modifications controlling protein activity. Smith et al. describe a strategy to add phosphorylation sites to globular proteins to regulate their functions using the Erbin PDZ domain as an example and propose using phosphoswitchable domains in synthetic biology.