Uncoupled substrate binding underlies the evolutionary switch between Na + and H + -coupled prokaryotic aspartate transporters.

Secondary active membrane transporters use the electrochemical energy of ion gradients to concentrate their substrates. Transporters within the same family often evolve to use different ions, driven by physiological needs or bioavailability. How such functional differences arise despite similar threedimensional protein structures is mostly unknown. We used phylogenetics and ancestral sequence reconstruction on prokaryotic glutamate transporters to recapitulate the evolutionary transition from Na + -coupled (GT-Na) to H + -coupled (GT-H) transport and discovered that it occurred via an intermediate clade (GT-Int). The reconstructed ancestral transporter AncGT-Int contains all residues required for Na + binding but switched from Na + -coupled substrate binding, characteristic of GT-Na transporters, to uncoupled binding. The high-resolution cryo-EM structures of AncGT-Int show that it binds substrate without Na + ions in the same manner as GT-Na transporters, which instead require Na + ions. Unlike GT-Na transporters, remodeled by ions into a high-affinity substrate-binding configuration, apo AncGT-Int is already in this configuration. Our results show how allosteric changes eliminated Na + dependence of Na + -coupled transporters before H + dependence arose, shedding light on ion coupling mechanisms and evolution in this family, and highlighting the power of phylogenetics and ancestral sequence reconstruction in the structure-function studies of membrane transporters.