Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes

Research output: Contribution to journalArticle

Authors

  • Ylva Ivarsson
  • Megan McLaughlin
  • Satra Nim
  • Rakesh Joshi
  • Debashish Ray
  • Bernard Liu
  • Joan Teyra
  • Tony Pawson
  • Jason Moffat
  • Shawn Shun-Cheng Li
  • Sachdev S Sidhu
  • Philip M Kim

Colleges, School and Institutes

External organisations

  • Department of Biochemistry, University of Regina, Regina, Saskatchewan S4S0A2, Canada, Banting and Best Department of Medical Research, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada, Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada, Department of Computer Science, University of Regina, Regina, Saskatchewan S4S0A2, Canada, Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, Department of Biology, Carleton University, Ottawa, Ontario K1S 5B6, Canada and Department of Computer Science, University of Toronto, Toronto, Ontario M5S 3G4, Canada Department of Biochemistry, University of Regina, Regina, Saskatchewan S4S0A2, Canada, Banting and Best Department of Medical Research, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada, Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada,

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.

Details

Original languageEnglish
Pages (from-to)2542-7
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume111
Issue number7
Publication statusPublished - 18 Feb 2014

Keywords

  • Bacteriophage M13, Computational Biology, DNA Primers, Humans, Microarray Analysis, PDZ Domains, Peptide Library, Protein Interaction Mapping, Proteomics, Journal Article, Research Support, Non-U.S. Gov't