• DOI:10.1073/pnas.1312296111
• Corpus ID: 3889698

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

@article{Ivarsson2014LargescaleIP,
  title={Large-scale interaction profiling of PDZ domains through proteomic peptide-phage display using human and viral phage peptidomes},
  author={Ylva Ivarsson and Roland Arnold and Megan E. McLaughlin and Satra Nim and Rakesh Joshi and Debashish Ray and Bernard A. Liu and Joan Teyra and Tony Pawson and Jason Moffat and Shawn S.-C. Li and Sachdev S. Sidhu and Philip M. Kim},
  journal={Proceedings of the National Academy of Sciences},
  year={2014},
  volume={111},
  pages={2542 - 2547}
}

• Y. Ivarsson, Roland Arnold, +10 authors Philip M. Kim
• Published 3 February 2014
• Biology
• Proceedings of the National Academy of Sciences

Significance Although knowledge about the human interactome is increasing in coverage because of the development of high-throughput technologies, fundamental gaps remain. In particular, interactions mediated by short linear motifs are of great importance for signaling, but systematic experimental approaches for their detection are missing. We fill this important gap by developing a dedicated approach that combines bioinformatics, custom oligonucleotide arrays and peptide-phage display. We computationally design a library of all possible motifs in a given proteome, print representatives of these on custom oligonucleotide arrays, and identify natural peptide binders for a given protein using phage display. Our approach is scalable and has broad application. Here, we present a proof-of-concept study using both designed human and viral peptide libraries. 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.