Conservation of Specificity in Two Low-Specificity Proteins

Abstract

Many regulatory proteins bind peptide regions of target proteins and modulate their activity. Such regulatory proteins can often interact with highly diverse target peptides. In many instances, it is not known if the peptide-binding interface discriminates targets in a biological context, or whether biological specificity is achieved exclusively through external factors such as subcellular localization. We used an evolutionary biochemical approach to distinguish these possibilities for two such low-specificity proteins: S100A5 and S100A6. We used isothermal titration calorimetry to study the binding of peptides with diverse sequence and biochemistry to human S100A5 and S100A6. These proteins bound distinct, but overlapping, sets of peptide targets. We then studied the peptide binding properties of orthologs sampled from across five amniote species. Binding specificity was conserved along all lineages, for the last 320 million years, despite the low specificity of each protein. We used ancestral sequence reconstruction to determine the binding specificity of the last common ancestor of the paralogs. The ancestor bound the entire set of peptides bound by modern S100A5 and S100A6 proteins, suggesting that paralog specificity evolved via subfunctionalization. To rule out the possibility that specificity is conserved because it is difficult to modify, we identified a single historical mutation that, when reverted in human S100A5, gave it the ability to bind an S100A6-specific peptide. These results reveal strong evolutionary constraints on peptide binding specificity. Despite being able to bind a large number of targets, the specificity of S100 peptide interfaces is likely important for the biology of these proteins.

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

“…The biochemically described S100 binding motifs, found in the literature, show an extremely low sequence similarity [15,23] (Fig 3A). Mostly linear segments are recognized by the human S100ome, however, no consensus S100 binding sequence can be defined [15]. In general, hydrophobic residues are preferred, but additional basic residues can also be favored in some instances.…”

“…Evidence for functional redundancy within the S100 family and possible functions of the orphan group S100 proteins are usually considered as 'sticky', relatively low specificity proteins [15], however no systematic study has been performed to make a specificity map involving the complete S100ome. Usually, the tested S100 proteins only covered the closest relatives (e.g.…”

“…Similarly to calmodulin, they can interact with protein or peptide targets in a calcium-dependent manner [14]. They are generally considered as relatively low specificity proteins, with dozens of interaction partners, among them they are unable to maintain a high selectivity [15]. In this study we determined the interaction profile of the full human S100 family (termed here as the S100ome) against a set of diverse known S100 partners (and some of their paralogs) systematically, including kinases such as RSK1 [16] and its paralogs MK2 and MNK1; cytoskeletal elements such as CapZ [17] (commonly known as TRTK12), NMIIA [18], ezrin [19], FOR20 and its paralog FOP [20]; membrane proteins such as NCX1 [15] and TRPM4 [21]; and other signaling proteins such as the tumor suppressor p53 [22][23][24], SIP [15] and MDM4 [23].…”

Divergent evolution of a protein-protein interaction revealed through ancestral sequence reconstruction and resurrection

“…Evolutionary biochemistry, i.e., phylogenetic reconstruction of ancestral sequences followed by expression and experimental characterization of the ancient proteins ( Fig. 2a-b) is a powerful tool for understanding protein function (Hochberg and Thornton 6 2017), including protein-protein interactions (Hultqvist et al 2017;Jemth et al 2018;Wheeler et al 2018). To better understand the evolution and hence function of the PDZ3:CRIPT interaction we reconstructed sequences of ancestral time-matched variants of PDZ3 and CRIPT and compared their binding affinity and stability to PDZ3 and CRIPT from seven extant species.…”

Learning peptide recognition rules for a low‐specificity protein

“…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. Specific targets within the highly-variable sets of S100 binding partners appear to be evolutionary conserved over hundreds of millions of years, even as the interface acquired mutations.…”