Evolution of multifunctionality through a pleiotropic substitution in the innate immune protein S100A9

Multifunctional proteins are evolutionary puzzles: how do proteins evolve to satisfy multiple functional constraints? S100A9 is one such multifunctional protein. It potently amplifies inflammation via Toll-like receptor 4 and is antimicrobial as part of a heterocomplex with S100A8. These two functions are seemingly regulated by proteolysis: S100A9 is readily degraded, while S100A8/S100A9 is resistant. We take an evolutionary biochemical approach to show that S100A9 evolved both functions and lost proteolytic resistance from a weakly proinflammatory, proteolytically resistant amniote ancestor. We identify a historical substitution that has pleiotropic effects on S100A9 proinflammatory activity and proteolytic resistance but has little effect on S100A8/S100A9 antimicrobial activity. We thus propose that mammals evolved S100A8/S100A9 antimicrobial and S100A9 proinflammatory activities concomitantly with a proteolytic “timer” to selectively regulate S100A9. This highlights how the same mutation can have pleiotropic effects on one functional state of a protein but not another, thus facilitating the evolution of multifunctionality.