The promiscuous functions of proteins are an important reservoir of functional
novelty in protein evolution, but the molecular basis for binding promiscuity
remains elusive. We used ancestral protein reconstruction to experimentally
characterize evolutionary intermediates in the functional expansion of the polar
amino acid-binding protein family, which has evolved to bind a variety of amino
acids with high affinity and specificity. High-resolution crystal structures of
an ancestral arginine-binding protein in complex with l-arginine and l-glutamine
show that the promiscuous binding of l-glutamine is enabled by multi-scale
conformational plasticity, water-mediated interactions, and selection of an
alternative conformational substate productive for l-glutamine binding.
Evolution of specialized glutamine-binding proteins from this ancestral protein
was achieved by displacement of water molecules from the protein-ligand
interface, reducing the entropic penalty associated with the promiscuous
interaction. These results provide a structural and thermodynamic basis for the
co-option of a promiscuous interaction in the evolution of binding specificity.
