Calprotectin is a multifunctional complex of S100 proteins A8 and A9. It promotes neutrophil migration to sites of inflammation, kills bacteria via oxidative bursting and transition metal sequestration, and exacerbates the inflammatory response by stimulating proinflammatory cell surface receptors. This extensive multifunctionality is the product of preferential heterocomplex formation between A8 and A9, as individual A8 or A9 homodimers are insufficient to perform all of these functions. Our research aims to understand how modern A8 and A9 proteins evolved from a shared homomeric ancestor to form the highly multifunctional calprotectin complex. When did A8/A9 heterocomplexation evolve? How have key biophysical differences between A8 and A9 homo‐ and heterocomplexes changed over time? What are the functional consequences of evolving preferential heterocomplexation? To answer these questions, we are examining complex formation between both extant and ancestrally reconstructed A8 and A9 proteins. We are also characterizing the biophysics of preferential heterocomplexation by measuring oligomeric state and proteolytic susceptibility for all members of the calgranulin clade (S100 proteins A8, A9, A12, and MRP126). Finally, we are mapping changes in biophysical properties and complex formation to changes in functionality. We have found that all therian A8s and A9s form proteolytically resistant heterocomplexes, while the ancestor of all calgranulins forms a proteolytically resistant homohexamer. Antimicrobial activity appears to be conserved across extant A8/A9 complexes, and we are currently evaluating this functionality for ancestral complexes. This work will help unravel the evolution of key biophysical features that contributed to the evolution of multifunctionality in this important innate immunity protein complex.Support or Funding InformationThis work was funded by the National Institutes of Health (NIH‐T32GM007413). Michael J. Harms is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.