Chapter 3
Identification of PDZ Interactions by Proteomic Peptide
Phage Display
Susanne Lu ¨ chow, Gustav N. Sundell, and Ylva Ivarsson
Abstract
PSD95-Disc large-Zonula occludens (PDZ) domains are among the most abundant modular domains in
the human proteome. They typically bind short carboxy-terminal sequence motifs of their ligand proteins,
which may be transmembrane proteins such as ion channels and GPCRs, as well as soluble proteins. The
identity of the endogenous ligands of many PDZ domains remains unclear despite more than two decades
of PDZ research. Combinatorial peptide phage display and bioinformatics predictions have contributed to
shed light on PDZ-mediated interactions. However, the efficiency of these methods for the identification of
interactions of potential biological relevance is hampered by different biases. Proteomic peptide-phage
display (ProP-PD) was developed to overcome these limitations. Here we describe a ProP-PD protocol for
the identification of C-terminal PDZ domain ligands. The method efficiently identifies peptide ligands
within a proteome of interest, and pinpoint targets of potential biological relevance.
Key words Phage display, PDZ domain, NGS, Specificity
1 Introduction
PDZ domains are abundant modular domains that are well-known
for binding to C-terminal binding motifs. The three main classes of
PDZ binding motifs are class I [T/S]-X-Φ-COO-, class II
Φ-X-Φ-COO-, and class III [D/E]-X-Φ-COO-, where X stands
for any amino acid and Φ stands for a hydrophobic amino acid
[1, 2]. The ligand binding specificities of a large portion of human
PDZ domains have been charted through combinatorial peptide
phage display [3], in which a highly diverse peptide phage display
library was used to identify high affinity ligands. The analysis
revealed that the specificities of PDZ domains may be expanded
and subdivided. The information has been used for large-scale
predictions of PDZ mediated interactions in the proteome [4],
and structures of representative cases of high-affinity interactions
have provided important insights into the binding determinant of
PDZ domain interactions [5]. The high-affinity binders generated
Jean-Paul Borg (ed.), PDZ Mediated Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 2256,
https://doi.org/10.1007/978-1-0716-1166-1_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
41
Identification of PDZ Interactions by Proteomic Peptide
Phage Display
Susanne Lu ¨ chow, Gustav N. Sundell, and Ylva Ivarsson
Abstract
PSD95-Disc large-Zonula occludens (PDZ) domains are among the most abundant modular domains in
the human proteome. They typically bind short carboxy-terminal sequence motifs of their ligand proteins,
which may be transmembrane proteins such as ion channels and GPCRs, as well as soluble proteins. The
identity of the endogenous ligands of many PDZ domains remains unclear despite more than two decades
of PDZ research. Combinatorial peptide phage display and bioinformatics predictions have contributed to
shed light on PDZ-mediated interactions. However, the efficiency of these methods for the identification of
interactions of potential biological relevance is hampered by different biases. Proteomic peptide-phage
display (ProP-PD) was developed to overcome these limitations. Here we describe a ProP-PD protocol for
the identification of C-terminal PDZ domain ligands. The method efficiently identifies peptide ligands
within a proteome of interest, and pinpoint targets of potential biological relevance.
Key words Phage display, PDZ domain, NGS, Specificity
1 Introduction
PDZ domains are abundant modular domains that are well-known
for binding to C-terminal binding motifs. The three main classes of
PDZ binding motifs are class I [T/S]-X-Φ-COO-, class II
Φ-X-Φ-COO-, and class III [D/E]-X-Φ-COO-, where X stands
for any amino acid and Φ stands for a hydrophobic amino acid
[1, 2]. The ligand binding specificities of a large portion of human
PDZ domains have been charted through combinatorial peptide
phage display [3], in which a highly diverse peptide phage display
library was used to identify high affinity ligands. The analysis
revealed that the specificities of PDZ domains may be expanded
and subdivided. The information has been used for large-scale
predictions of PDZ mediated interactions in the proteome [4],
and structures of representative cases of high-affinity interactions
have provided important insights into the binding determinant of
PDZ domain interactions [5]. The high-affinity binders generated
Jean-Paul Borg (ed.), PDZ Mediated Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 2256,
https://doi.org/10.1007/978-1-0716-1166-1_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
41