The melibiose carrier from Escherichia coli (MelB) couples the accumulation of the disaccharide melibiose to the downhill entry of H(+), Na(+), or Li(+). In this work, substrate-induced FTIR difference spectroscopy was used in combination with fluorescence spectroscopy to quantitatively compare the conformational properties of MelB mutants, implicated previously in sodium binding, with those of a fully functional Cys-less MelB permease. The results first suggest that Asp55 and Asp59 are essential ligands for Na(+) binding. Secondly, though Asp124 is not essential for Na(+) binding, this acidic residue may play a critical role, possibly by its interaction with the bound cation, in the full Na(+)-induced conformational changes required for efficient coupling between the ion- and sugar-binding sites; this residue may also be a sugar ligand. Thirdly, Asp19 does not participate in Na(+) binding but it is a melibiose ligand. The location of these residues in two independent threading models of MelB is consistent with their proposed role.