title: Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes.
abstract: The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain-SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host-virus protein-protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen-host protein-protein interactions.
authors.affiliation: Department of Molecular Genetics, Department of Computer Science, Donnelly Centre, University of Toronto, Toronto, ON, Canada M5S 3E1.
authors.forename: Ylva
authors.initials: Y
authors.lastname: Ivarsson
authors.forename: Roland
authors.initials: R
authors.lastname: Arnold
authors.forename: Megan
authors.initials: M
authors.lastname: McLaughlin
authors.forename: Satra
authors.initials: S
authors.lastname: Nim
authors.forename: Rakesh
authors.initials: R
authors.lastname: Joshi
authors.forename: Debashish
authors.initials: D
authors.lastname: Ray
authors.forename: Bernard
authors.initials: B
authors.lastname: Liu
authors.forename: Joan
authors.initials: J
authors.lastname: Teyra
authors.forename: Tony
authors.initials: T
authors.lastname: Pawson
authors.forename: Jason
authors.initials: J
authors.lastname: Moffat
authors.forename: Shawn Shun-Cheng
authors.initials: SS
authors.lastname: Li
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.forename: Philip M
authors.initials: PM
authors.lastname: Kim
mesh_terms: D017104:Bacteriophage M13 / Q000235:genetics*; D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics; D006801:Humans; D046228:Microarray Analysis; D054731:PDZ Domains / Q000235:genetics* / Q000502:physiology*; D019151:Peptide Library; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*
chemical_list: D017931:DNA Primers; D019151:Peptide Library