Ancestral protein reconstruction allows the resurrection and characterization of ancient proteins based on computational analyses
of sequences of modern-day proteins. Unfortunately, many protein families are highly divergent and not suitable for sequence-based
reconstruction approaches. This limitation is exemplified by the antigen receptors of jawed vertebrates (B- and T-cell receptors),
heterodimers formed by pairs of Ig domains. These receptors are believed to have evolved from an extinct homodimeric ancestor
through a process of gene duplication and diversification; however molecular evidence has so far remained elusive. Here, we
use a structural approach and laboratory evolution to reconstruct such molecules and characterize their interaction with antigen.
High-resolution crystal structures of reconstructed homodimeric receptors in complex with hen-egg white lysozyme demonstrate
how nanomolar affinity binding of asymmetrical antigen is enabled through selective recruitment and structural plasticity
within the receptor-binding site. Our results provide structural evidence in support of long-held theories concerning the
evolution of antigen receptors, and provide a blueprint for the experimental reconstruction of protein ancestry in the absence
of phylogenetic evidence.