Interactomic affinity profiling by holdup assay: acetylation and distal residues impact the PDZome-binding specificity of PTEN phosphatase

Abstract

<jats:title>Abstract</jats:title><jats:p>Protein domains often recognize short linear protein motifs composed of a core conserved consensus sequence surrounded by less critical, modulatory positions. Here we used an accurate experimental approach combining high-throughput holdup chromatographic assay and fluorescence polarization to measure quantitative binding affinity profiles of the PDZ domain-binding motif (PBM) of PTEN phosphatase towards the 266 known human PDZ domains. Inclusion of N-terminal flanking residues, acetylation or mutation of a lysine at a modulatory position significantly altered the PDZome-binding profile of the PTEN PBM. A specificity index is also introduced to quantify the specificity of a given PBM over the complete PDZome. Our results highlight the impact of modulatory residues and post-translational modifications on PBM interactomes and their specificity.</jats:p>

Host PDZ-containing proteins targeted by SARS-Cov-2

“…Immobilized peptide concentrations were calculated using measured dissociation constants from competitive fluorescent polarization assays (see below). We have found that all determined BI-Kd pairs resulted a mean peptide concentration of ~20 μM, a concentration that is coherent with other peptides in the same method [22,23,60]. The minimal Binding Intensity (BI) threshold value is 0.2 to define a significant interaction, roughly corresponding to a 100 μM dissociation constant as previously reported [19].…”

“…Each PDZ of the library is defined by the protein name followed by the PDZ number in the protein. The holdup approach displays a high sensitivity of detection for low-to-medium affinity PDZ/PBM pairs and provides an affinity-based ranking of the identified binders corresponding to a specificity profile [22]. The specificity profiles for SARS-CoV, SARS-CoV-2 and MERS-CoV E PBM with the mean values of binding intensities (BI) are reported in Fig.…”

Quantitative fragmentomics allow affinity mapping of interactomes

“…To explore the PDZ-PBM interactome, we expressed a recombinant PDZome library covering all the 266 known human PDZs (Duhoo, Girault et al, 2019), and we synthetized a 10-mer peptide library of 24 viral and 424 human PBMs, of which 323, 63, 51, and 11 belong to class 1, 2, 3, and to atypical or non-C-terminal subgroups, respectively (Table S1). 8 PBMs harboured post-translational modifications (phosphorylation or acetylation) which may modulate binding specificities (Gógl et al, 2019) (Gogl et al, 2020) (Jané et al, 2020). The viral PBMs included notably 12 PBMs from oncoproteins HPV E6 (11 distinct types) and HTLV1 TAX1.…”

“…To quantify dissociation constants, we used the holdup method, a high-throughput comparative chromatographic retention assay we developed previously (Charbonnier et al, 2006) (Vincentelli et al, 2015) (Jané et al, 2020). The assay measures the total and unbound concentrations of reactants at equilibrium, which can be converted into steady-state dissociation constants herein reported as pK d values (the negative of the base 10 logarithm of the dissociation constant).…”

“…For any individual PDZ or PBM measured in our quantitative interactome, binding specificities can be visualized by plotting binding profiles with pK d values sorted in decreasing order (Figure 1, upper right panel or Figure 3A,D). The steeper the profile slope, the more specific the PBM or PDZ for particular targets within the explored interactome (Vincentelli et al, 2015) (Jané et al, 2020). The PBM-binding preferences of PDZ domains can also be visualized by calculating affinity-weighted sequence logos (see Methods).…”