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

• Published in: The FEBS journal 2020-07, Vol.287 (13), p.2834-2846
• Main Authors: Simon, Márton A., Ecsédi, Péter, Kovács, Gábor M., Póti, Ádám L., Reményi, Attila, Kardos, József, Gógl, Gergő, Nyitray, László
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Assaying; Binding, Competitive; Biochemistry, Molecular Biology; Calcium; Fluorescence; fluorescence anisotropy; Fluorescence polarization; Fluorescence Polarization - methods; High-Throughput Screening Assays - methods; Humans; isothermal titration calorimetry; Life Sciences; Ligands; Phylogeny; Polarization; Protein Binding; Protein families; Protein interaction; Protein Interaction Domains and Motifs; Proteins; Redundancy; S100 protein; S100 Proteins - metabolism; Sequence Homology; systems biology; Vertebrates
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.15175

The calcium‐binding, vertebrate‐specific S100 protein family consists of 20 paralogs in humans (referred as the S100ome), with several clinically important members. To explore their protein–protein interactions (PPIs) quantitatively, we have chosen an unbiased, high‐throughput, competitive fluorescence polarization (FP) assay 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. Our results demonstrate that FP assays are highly suitable for quantitative interaction profiling of selected protein families. Moreover, we provide evidence that PPI‐based phenotypic characterization can complement or even exceed the information obtained from the sequence‐based phylogenetic analysis of the S100ome, an evolutionary young protein family. The calcium‐binding dimeric S100 protein family can be separated into two groups based on their specificity profile against a large set of interaction partners. The minor group contains promiscuous S100 proteins with a clear sign of functional redundancy, while the larger group consists of S100 members without a clear binding preference (orphan).