Chapter 4
A Computational Protocol to Analyze PDZ/PBM Affinity Data
Obtained by High-Throughput Holdup Assay
Pau Jane ´ , Lionel Chiron, Goran Bich, Gilles Trave ´ , and Yves Nomine ´
Abstract
The holdup assay is an automated high-throughput comparative chromatographic retention approach that
allows to measure quantitative binding intensities (BI) for a large number of domain–motif pairs and
deduce equilibrium binding affinity constants. We routinely apply this approach to obtain quantitative
binding specificity profiles of particular PDZ-binding motifs (PBMs) toward the full library of known
human PDZ domains (the PDZome). The quality of the electropherograms extracted from the capillary
electrophoresis instrument at the final step of the holdup assay may vary, influencing the accuracy and
reproducibility of the measurement. By using bioinformatic tools, we can solve these issues to extract more
reliable BIs by means of a better superimposition of the electropherograms. The protocol presented in this
chapter describes the main principles and strategies of our curated method to process holdup data and new
ways to plot and compare the BIs for the PBM–PDZ interactions. For this particular protocol, all the
necessary computing commands are freely available in open Python packages.
Key words Holdup assay, PDZ–PBM interaction, Computational approach, Processing accuracy,
Electropherogram superimposition
1 Introduction
An important subset of protein–protein interactions are mediated
by globular protein domains interacting with short conserved linear
motifs (SLiMs), mostly belonging to intrinsically unfolded regions
of the proteome. Many high-throughput interatomic data allow to
describe protein–SLiM interactions only in a binary way (“binds”
or “does not bind”) [1–4]. The quantification of the binding
affinities would lead to a better understanding of the hierarchies
and specificities of interactions.
In this regard, the holdup assay [5–7] is well suited to extract
quantitative affinity information for an entire network of domain–
SLiM interactions, such as the 266 known human PDZ domains,
and their target motifs, called PDZ-Binding Motifs (PBM). The
holdup assay is a comparative chromatographic retention approach
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_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021
61
A Computational Protocol to Analyze PDZ/PBM Affinity Data
Obtained by High-Throughput Holdup Assay
Pau Jane ´ , Lionel Chiron, Goran Bich, Gilles Trave ´ , and Yves Nomine ´
Abstract
The holdup assay is an automated high-throughput comparative chromatographic retention approach that
allows to measure quantitative binding intensities (BI) for a large number of domain–motif pairs and
deduce equilibrium binding affinity constants. We routinely apply this approach to obtain quantitative
binding specificity profiles of particular PDZ-binding motifs (PBMs) toward the full library of known
human PDZ domains (the PDZome). The quality of the electropherograms extracted from the capillary
electrophoresis instrument at the final step of the holdup assay may vary, influencing the accuracy and
reproducibility of the measurement. By using bioinformatic tools, we can solve these issues to extract more
reliable BIs by means of a better superimposition of the electropherograms. The protocol presented in this
chapter describes the main principles and strategies of our curated method to process holdup data and new
ways to plot and compare the BIs for the PBM–PDZ interactions. For this particular protocol, all the
necessary computing commands are freely available in open Python packages.
Key words Holdup assay, PDZ–PBM interaction, Computational approach, Processing accuracy,
Electropherogram superimposition
1 Introduction
An important subset of protein–protein interactions are mediated
by globular protein domains interacting with short conserved linear
motifs (SLiMs), mostly belonging to intrinsically unfolded regions
of the proteome. Many high-throughput interatomic data allow to
describe protein–SLiM interactions only in a binary way (“binds”
or “does not bind”) [1–4]. The quantification of the binding
affinities would lead to a better understanding of the hierarchies
and specificities of interactions.
In this regard, the holdup assay [5–7] is well suited to extract
quantitative affinity information for an entire network of domain–
SLiM interactions, such as the 266 known human PDZ domains,
and their target motifs, called PDZ-Binding Motifs (PBM). The
holdup assay is a comparative chromatographic retention approach
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_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021
61