The PDZ domain is a protein–protein interacting module that plays an important role in the organization of signaling complexes. The recognition of short intrinsically disordered C‐terminal peptide motifs is the archetypical PDZ function, but the functional repertoire of this versatile module also includes recognition of internal peptide sequences, dimerization and phospholipid binding. The PDZ function can be tuned by various means such as allosteric effects, changes of physiological buffer conditions and phosphorylation of PDZ domains and/or ligands, which poses PDZ domains as dynamic regulators of cell signaling. This review is focused on the plasticity of the PDZ interactions.

Significance: Although knowledge about the human interactome is increasing in coverage because of the development of high-throughput technologies, fundamental gaps remain. In particular, interactions mediated by short linear motifs are of great importance for signaling, but systematic experimental approaches for their detection are missing. We fill this important gap by developing a dedicated approach that combines bioinformatics, custom oligonucleotide arrays and peptide-phage display. We computationally design a library of all possible motifs in a given proteome, print representatives of these on custom oligonucleotide arrays, and identify natural peptide binders for a given protein using phage display. Our approach is scalable and has broad application. Here, we present a proof-of-concept study using both designed human and viral peptide libraries.

Evolution of protein function can be driven by positive selection of advantageous nonsynonymous codon mutations that arise following gene duplication. By observing the presence and degree of site-specific positive selection for change between divergent paralogs, residue positions responsible for functional changes can be identified. We applied this analysis to genes encoding Mu class glutathione transferases, which differ widely in substrate specificities. Approximately 3% of the amino acid residue positions, both near to and distant from the active site, are under statistically significant positive selection for change. Relevant human glutathione transferase (GST) M1-1 and GST M2-2 codons were mutated. A chemically conservative threonine to serine mutation in GST M2-2 elicited a 1,000-fold increase in specific activity with the GST M1-1-specific substrate trans-stilbene oxide and a 30-fold increase with the alternative epoxide substrates styrene oxide and nitrophenyl glycidol. The reverse mutation in GST M1-1 resulted in reciprocal decreases in activity. Thus, identification of hypervariable codon positions can be a powerful aid in the redesign of protein function, lessening the requirement for extensive mutagenesis or structural knowledge and sometimes suggesting mutations that would otherwise be considered functionally conservative.

The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide‐binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large‐scale identification. We present a novel proteomic peptide phage display (ProP‐PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large‐scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP‐PD library was validated through selections against SLiM‐binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM‐binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non‐C‐terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein–protein interactions (PPIs) were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40–130 μm through isothermal titration calorimetry, and confirmed interactions through coimmunoprecipitation using full‐length proteins. Taken together, we outline a general pipeline for the design and construction of ProP‐PD libraries and the analysis of ProP‐PD‐derived, SLiM‐based PPIs. We demonstrated the methods potential to identify low affinity motif‐mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for PPI discovery.