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

Authors

Ylva Ivarsson, University of Toronto Faculty of Medicine
Roland Arnold, University of Toronto Faculty of Medicine
Megan McLaughlin, University of Toronto Faculty of Medicine
Satra Nim, University of Toronto Faculty of Medicine
Rakesh Joshi, Western University
Debashish Ray, University of Toronto Faculty of Medicine
Bernard Liu, Mount Sinai Hospital of University of Toronto
Joan Teyra, University of Toronto Faculty of Medicine
Tony Seitova, University of Toronto
Ashley Hutchinson, University of Toronto
Farhad Yusifov, University of Toronto
Tatiana Skarina, University of Toronto
Elena Evdokimova, University of Toronto
Peter Loppnau, University of Toronto
Pegah Ghiabi, University of Toronto
Taraneh Haijan, University of Toronto
Shanshan Zhong, Schulich School of Medicine & Dentistry
Husam Abdoh, Schulich School of Medicine & Dentistry
Benjamin D. Hedley, Schulich School of Medicine & Dentistry
Vipin Bhayana, Schulich School of Medicine & Dentistry
Claudio M. Martin, Western University
Marat Slessarev, Western University
Benjamin Chin-Yee, Schulich School of Medicine & Dentistry
Douglas D. Fraser, Western University
Ian Chin-Yee, Schulich School of Medicine & Dentistry
Shawn S.C. Li, Schulich School of Medicine & DentistryFollow

Document Type

Article

Publication Date

2-18-2014

Journal

Proceedings of the National Academy of Sciences of the United States of America

Volume

111

Issue

7

First Page

2542

Last Page

2547

URL with Digital Object Identifier

10.1073/pnas.1312296111

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 fulllength Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit a-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.