Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

Abstract

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from Human Papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human PDZome. We obtained exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes.

Rewiring of RSK-PDZ interactions by linear motif phosphorylation

“…Holdup assay The automated holdup assay was carried out against peptides (RSK1 725-735 ) in triplicates as previously described (Vincentelli et al, 2015) with minor modifications. For the detailed protocol please look at (Duhoo et al).…”

“…To investigate how phosphorylation can modulate the binding of the PBM of RSK to PDZ domains, we used the automated high-throughput holdup assay, which allows to measure binding intensities (BI values) for large numbers of domain-motif pairs experimentally. As compared to the original work describing this approach (Vincentelli et al, 2015), we used an updated version of our PDZ library, which now includes the 266 known human PDZ domains (Duhoo et al). Here, out of 266 PDZ, we could quantify the interaction of 255 PDZ for the unphosphorylated RSK1 peptide and 252 for the phosphorylated form.…”

“…A second challenge would be to use a quantitative method based on binding affinity measurements, rather than a simplistic binary description of the binding mode ("bind" or "do not bind"). Indeed, a recently developed "holdup assay" allows one to measure the individual binding affinities of the 266 known human PDZ domains for a single PBM, thereby generating a "quantitative PDZome-binding profile" of the motif (Vincentelli et al, 2015). Here, we applied this holdup assay method to generate the PDZome-binding profiles of both the unphosphorylated and phosphorylated RSK1 PBMs.…”

High throughput competitive fluorescence polarization assay reveals functional redundancy in the S100 protein family

“…The accurate determination of thermodynamic parameters of molecular interactions is performed by fast, but superficial high-throughput (HTP) methods. In the literature several HTP approaches are applied such as co-immunoprecipitation [2], yeast two hybrid and spot assays [3], pull-down assay [4], holdup assay [5] and direct fluorescence polarization/anisotropy [6]. In direct fluorescence polarization (FP) experiments, a fluorescent probe (usually a labeled peptide) is titrated with a globular partner.…”

Specificity in PDZ-peptide interaction networks: Computational analysis and review

“…On a PDZome-wide scale, the holdup assay provides a high-throughput technique to quantitate peptide binding to almost the full complement of human PDZ domains. This experimental assay uses microfluidic capillary electrophoresis to measure binding affinities of 266 recombinantly produced human PDZ domains, as well as 87 tandem domains, with peptide-coated resins, measuring up to 1000 binding affinities per day (Duhoo et al, 2019;Vincentelli et al, 2015).…”