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

Abstract

The calcium-binding, vertebrate-specific S100 protein family consists of 20 paralogs in humans (referred as the S100ome), with several clinically important members. To assess their interactome, high-throughput, systematic analysis is indispensable, which allows one to get not only qualitative but quantitative insight into their protein-protein interactions (PPIs). We have chosen an unbiased assay, fluorescence polarization (FP) that revealed a partial functional redundancy when the complete S100ome (n=20) was tested against numerous model partners (n=13). Based on their specificity, the S100ome can be grouped into two distinct classes: promiscuous and orphan. In the first group, members bound to several ligands (>4-5) with comparable high affinity, while in the second one, the paralogs bound only one partner weakly, or no ligand was identified (orphan). Our results demonstrate that in vitro FP assays are highly suitable for quantitative ligand binding studies of selected protein families. Moreover, we provide evidence that PPI-based phenotypic characterization can complement the information obtained from the sequence-based phylogenetic analysis of the S100ome, an evolutionary young protein family. Author summaryFunctional similarity among a protein family can be essential in order to understand proteomic data, to find biomarkers, or in inhibitor design. Proteins with similar functions can compensate the loss-offunction of the others, their expression can co-vary under pathological conditions, and simultaneous targeting can lead to better results in the clinics. To investigate this property one can use sequencebased approaches. However, this path can be difficult. In the case of the vertebrate specific, evolutionary young, S100 family, phylogenetic approaches lead to ambiguous results. To overcome this problem, we applied a high-throughput biochemical approach to experimentally measure the binding affinities of a large number of S100 interactions. We performed unbiased fluorescence polarization assay, involving the complete human S100ome (20 paralogs) and 13 known interaction partners. We used this measured 20x13 (260) protein-protein interaction array to reveal the functional relationships within the family. Our work provide a general framework for studies focusing on phenotype-based domain classification.

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

“…Direct and competitive titration experiments were carried out in triplicate. The average competitive FP signal was used for fitting the data to a competitive binding equation with ProFit, an in-house Python-based program [41], allowing to extract the apparent affinity values. In our FP assays, every tested PDZ domain detectably bound to at least one PBM peptide, guarantying that PDZ domains are functional.…”

“…All those normalization and alignment steps are performed automatically and are critical as the overlap of the two electropherograms is never perfect. be labeled, and minimizes the detection of false-positive partners, as compared to direct FP measurements [41]. The PBM peptide tracers to be labeled (f16E6, fRSK1 and fpRSK1) were picked from our PDZ/PBM data base so that each of the 20 PDZ present in the subset was targeted by at least one of those three peptides.…”

Learning peptide recognition rules for a low‐specificity protein

“…The protein has a very tight expression pattern and appears to be localized near specific bilayers, 45–47 thus limiting its available binding targets. hA5 also has relatively low affinities for peptides (≈ μM), 19,20 meaning that both it and/or its partners must be at relatively high concentration for an interaction to form. Finally, it is also possible that there are additional higher‐ordered properties of proteins that restrict the true set of possible hA5 target peptides.…”

“… 10–18 Peptides can interact with a single monomer in the homodimer (Figure 1b,c), or can interact across the homodimer interface (Figure 1d–f). Defining the peptide recognition rules of S100 proteins has, however, proven extremely difficult, as target peptides lack sufficient similarity to be usefully represented as a binding motif 19,20 . That said, the low‐specificity of the S100 protein family does not equate to no specificity .…”

“…This protein likely plays a signaling role in olfaction 21,22 . It interacts with a diverse set of peptide targets with no obvious sequence motif 18–20 . Instead of representing hA5's peptide specificity with a binding motif, we represented it using readily calculated biochemical properties of each peptide.…”