Proteomic peptide phage display uncovers novel interactions of the PDZ1‐2 supramodule of syntenin

Abstract

Syntenin has crucial roles in cell adhesion, cell migration and synaptic transmission. Its closely linked postsynaptic density‐95, discs large 1, zonula occludens‐1 (PDZ) domains typically interact with C‐terminal ligands. We profile syntenin PDZ1‐2 through proteomic peptide phage display (ProP‐PD) using a library that displays C‐terminal regions of the human proteome. The protein recognizes a broad range of peptides, with a preference for hydrophobic motifs and has a tendency to recognize cryptic internal ligands. We validate the interaction with nectin‐1 through orthogonal assays. The study demonstrates the power of ProP‐PD as a complementary approach to uncover interactions of potential biological relevance.

Proteome-wide analysis of phospho-regulated PDZ domain interactions through phosphomimetic proteomic peptide phage display

“…This region was not determined in the free Scribble PDZ1 domain, perhaps because the sequence used for the structure determination was shortened both at the C-and N-termini (PDB codes 1X5Q, 2W4F). However, we did not see any 15 N-1 H chemical shift difference in the HSQC spectra of the amino acid residues comprising this region between the free and bound protein. This suggests that the extra C-terminal structure is likely present in the free protein too, and might function as an extra scaffold for stability.…”

“…14 . Binding-enriched phage pools were analyzed by NGS, which provided information on the binding peptides from which the full-length proteins could be identified 12,15 . In addition, the analysis provided an affinity ranked list of ligands as the differences observed in NGS counts for peptides identified within a given selection typically translate into affinity differences.…”

PDZ Domains as Drug Targets

“…[55] As PIP2 is not present in some cellular compartments, this would favor specific cellular locations of syntenin. [251] The best-characterized phage display hit is the heptamer SKKEWYV peptide (Figure 11h). The first smallmolecule syntenin inhibitor, 113B7 (Figure 11h), was developed by fragment-based drug design by NMR screening of an in-house assembled fragment library comprising about 5000 compounds against syntenin PDZ12.…”

“…[243] Syntenin has a unique function of facilitating both intracellular and extracellular events of invasion and has thus evolved as a promising target for cancer intervention. [251] [250] Two fragments were identified to mainly interact with the PDZ1 domain and within the interface between the two PDZ domains.…”

High‐throughput discovery of functional disordered regions: investigation of transactivation domains

“…In vitro technologies such as phage display are powerful to identify short disordered linear motifs (three to seven residues within IDRs) that can mediate interactions with specific protein domains in vitro as well as discover strong binders (Ivarsson et al , ; Garrido‐Urbani et al , ; Davey et al , ). Such approaches require screening short peptides against specific interaction partners, thus constraining the mechanism by which they mediate function (Jones et al , ; Ivarsson et al , ; Garrido‐Urbani et al , ; Davey et al , ). The screening occurs outside of the relevant cellular/biological context during the selection experiment and hence does not explicitly consider cellular specificity for binding, i.e., selection against promiscuous binding with other molecules in the cell (negative selection).…”