Multiple Evolutionary Origins of Ubiquitous Cu2+ and Zn2+ Binding in the S100 Protein Family

Abstract

The S100 proteins are a large family of signaling proteins that play critical roles in biology and disease. Many S100 proteins bind Zn2+, Cu2+, and/or Mn2+ as part of their biological functions; however, the evolutionary origins of binding remain obscure. One key question is whether divalent transition metal binding is ancestral, or instead arose independently on multiple lineages. To tackle this question, we combined phylogenetics with biophysical characterization of modern S100 proteins. We demonstrate an earlier origin for established S100 subfamilies than previously believed, and reveal that transition metal binding is widely distributed across the tree. Using isothermal titration calorimetry, we found that Cu2+ and Zn2+ binding are common features of the family: the full breadth of human S100 paralogs—as well as two early-branching S100 proteins found in the tunicate Oikopleura dioica—bind these metals with μM affinity and stoichiometries ranging from 1:1 to 3:1 (metal:protein). While binding is consistent across the tree, structural responses to binding are quite variable. Further, mutational analysis and structural modeling revealed that transition metal binding occurs at different sites in different S100 proteins. This is consistent with multiple origins of transition metal binding over the evolution of this protein family. Our work reveals an evolutionary pattern in which the overall phenotype of binding is a constant feature of S100 proteins, even while the site and mechanism of binding is evolutionarily labile.

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

“…The phylogenetic analyses of the human S100ome resulted in rather ambiguous genealogies, likely due to the young age of the protein family (Fig S17.). Nevertheless, the clade including S100A2, S100A3, S100A4, S100A5, S100A6 was supported with high statistical values in all analyses ( Fig S17.), similarly as it had also been found by others [29,30]. Our functional analysis has revealed that all members of this clade belong to the same subset of the promiscuous group, with a greatly similar functional profile.…”

“…It is a Chordata-specific, evolutionary young protein family, and despite the fact that they exhibit moderate sequence similarity, they are structurally very similar owing to their small size (~100 residues) and conserved fold (including two consecutive EF hand motifs) ( Fig S16.). Due to this reason, their phylogenetic analysis generally does not lead to unambiguous results [29,30]. Applying different parameters during the analyses resulted in varied grouping of the human S100ome, moreover only a few clades received statistical supports (see our analyses in Fig S17.).…”

“…Peptide synthesis. The CapZ (265-276), NCX1 (254-265), SIP (188-202), TRPM4 (129-147) and MDM4(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43) peptides were chemically synthesized using solid phase peptide synthesis (PS3 peptide synthesizer, Protein Technologies) with Fmoc/tBu strategy in the case of (5(6)-carboxyfluorescein) labeled and unlabeled version. Peptides were purified by RP-HPLC using a Jupiter 300 Å C 18 column (Phenomenex).…”

En kvinne med residiverende feber, tørrhoste og ascites

“…Metoden brukes for å påvise spesifikke proteiner i cellen eller på cellemembranen, og er viktig innenfor diagnostikken av cancer, infeksjonssykdom og immunologisk sykdom. CD68 uttrykkes av monocytter og vevsmakrofager (12), CD1a brukes ofte som kjennetegn på dendrittiske celler (13), mens S100 utgjør en gruppe proteiner som er viktige innenfor inflammatoriske sykdommer, kreft og immunforsvaret (14).…”

“…Dyp venetrombose ble bekreftet med ultralydundersøkelse, og det ble samtidig funnet trombocytemi med en trombocyttverdi på 1026 · 10 9 /l (145-390). Hun hadde i tillegg leukocytose med leukocyttverdi på 12,3 · 10 9 /l (3,7-10,0), mens hemoglobinnivået var 13,8 g/100 ml (11,(7)(8)(9)(10)(11)(12)(13)(14)(15)3). Videre utredning påviste JAK2-V617F-mutasjon, og diagnosen essensiell trombocytose ble stilt.…”