biostudies-literatureLongo LMHuman Frontier Science ProgramIsrael Science FoundationKnut och Alice Wallenbergs Stiftelse5310-5318https://www.ebi.ac.uk/biostudies/studies/S-EPMC7071883biostudies-literatureUnknown117(10)The ubiquity of phospho-ligands suggests that phosphate binding emerged at the earliest stage of protein evolution. To evaluate this hypothesis and unravel its details, we identified all phosphate-binding protein lineages in the Evolutionary Classification of Protein Domains database. We found at least 250 independent evolutionary lineages that bind small molecule cofactors and metabolites with phosphate moieties. For many lineages, phosphate binding emerged later as a niche functionality, but for the oldest protein lineages, phosphate binding was the founding function. Across some 4 billion y of protein evolution, side-chain binding, in which the phosphate moiety does not interact with the backbone at all, emerged most frequently. However, in the oldest lineages, and most characteristicalProceedings of the National Academy of Sciences of the United States of AmericaShort and simple sequences favored the emergence of N-helix phospho-ligand binding sites in the first enzymes.PMC70718832018.01402013.0124980/14RGP0041/2017Kamerlin SCLTawfik DSPetrovic DLongo LMfalseShort and simple sequences favored the emergence of N-helix phospho-ligand binding sites in the first enzymes.The ubiquity of phospho-ligands suggests that phosphate binding emerged at the earliest stage of protein evolution. To evaluate this hypothesis and unravel its details, we identified all phosphate-binding protein lineages in the Evolutionary Classification of Protein Domains database. We found at least 250 independent evolutionary lineages that bind small molecule cofactors and metabolites with phosphate moieties. For many lineages, phosphate binding emerged later as a niche functionality, but for the oldest protein lineages, phosphate binding was the founding function. Across some 4 billion y of protein evolution, side-chain binding, in which the phosphate moiety does not interact with the backbone at all, emerged most frequently. However, in the oldest lineages, and most characteristical2020-01-01T00:00:00Z2020 Mar2026-05-02T06:25:23.113Z2026-04-07T17:41:27.853ZS-EPMC70718833207972210.1073/pnas.1911742117