New binding specificities evolve via point mutation in an invertebrate allorecognition gene.

Many organisms use genetic self-recognition systems to distinguish themselves from conspecifics. In the cnidarian, , self-recognition is partially controlled by (). encodes a highly polymorphic transmembrane protein that discriminates self from nonself by binding to other Alr2 proteins with identical or similar sequences. Here, we focused on the N-terminal domain of Alr2, which can determine its binding specificity. We pair ancestral sequence reconstruction and experimental assays to show that amino acid substitutions can create sequences with novel binding specificities either directly (via one mutation) or via sequential mutations and intermediates with relaxed specificities. We also show that one side of the domain has experienced positive selection and likely forms the binding interface. Our results provide direct evidence that point mutations can generate Alr2 proteins with novel binding specificities. This provides a plausible mechanism for the generation and maintenance of functional variation in nature.