Using peptide-phage display to capture conditional motif-based interactions
- Plats: B42, BMC, Husargatan 3, Uppsala
- Doktorand: Sundell, Gustav
- Om avhandlingen
- Arrangör: Biokemi
- Kontaktperson: Sundell, Gustav
This thesis explores the world of conditional protein-protein interactions using combinatorial peptide-phage display and proteomic peptide-phage display (ProP-PD). Large parts of proteins in the human proteome do not fold in to well-defined structures instead they are intrinsically disordered. The disordered parts are enriched in linear binding-motifs that participate in protein-protein interaction. These motifs are 3-12 residue long stretches of proteins where post-translational modifications, like protein phosphorylation, can occur changing the binding preference of the motif. Allosteric changes in a protein or domain due to phosphorylation or binding to second messenger molecules like Ca2+ can also lead conditional interactions. Finding phosphorylation regulated motif-based interactions on a proteome-wide scale has been a challenge for the scientific community.
Through combinatorial peptide phage display selections against the calcium binding protein Sorcin we discovered the Φ-Φ-X-P motif that preferentially interacts with calcium bound Sorcin. Through C-terminal ProP-PD we discovered that the engineered L391F, K329M Ø9 mutant of the second PDZ domain of DLG1 has a reduced specificity and increased affinity towards proteomic targets as compared to the wild type DLG1 PDZ2 domain.
Finally a novel application of ProP-PD that allows us to capture phospho-regulated interactions by combining it with glutamic phosphomimetic substitution is presented. We created a phosphomimetic ProP-PD library of C-terminal sequences of known and putative serine/threonine phospho-sites, and their phosphomimetic counterparts. The library was used in selection against PDZ 1, 2 and 3 of Scribble and DLG1. We found site-specific phosphorylation events that either enable or disable interactions. Selectivity towards phosphopeptide binding was confirmed for Scribble PDZ1 through biophysical experiments, supporting the viability of the phosphomimetic ProP-PD. Through structural and mutational analysis we found that the gate-keeping residue for phosphopeptide binding in Scribble PDZ1 is arginine 762. With this proof-of-concept study we have introduced phosphomimetic ProP-PD as a viable method to discover phospho-regulated protein-peptide interactions.
Taken together, this work has contributed with methods to capture selectivity differences in motif-based interactions of proteins depending on allosteric binding of calcium, protein engineering and on ligand modification by phosphorylation.