LAUSR

Toll‐like receptor 4 (TLR4) is a critical innate immune protein that activates inflammation in response to extracellular cues. Much of the work to understand how the protein works in humans has been done using mouse models. Although human and mouse TLR4 have many shared features, they have also diverged significantly since their last common ancestor, acquiring 277 sequence differences. Functional differences include the extent of ligand‐independent activation, whether lipid IVa acts as an antagonist or agonist, and the relative species cross‐compatibility of their MD‐2 cofactor. We set out to understand the evolutionary origins for these functional differences between human and mouse TLR4. Using a combination of phylogenetics, ancestral sequence reconstruction, and functional characterization, we found that evolutionary changes to the human TLR4, rather than changes to the mouse TLR4, were largely responsible for these functional changes. Human TLR4 repressed ancestral ligand‐independent activity and gained antagonism to lipid IVa. Additionally, mutations to the human TLR4 cofactor MD‐2 led to lineage‐specific incompatibility between human and opossum TLR4 complex members. These results were surprising, as mouse TLR4 has acquired many more mutations than human TLR4 since their last common ancestor. Our work has polarized this set of transitions and sets up work to study the mechanistic underpinnings for the evolution of new functions in TLR4.