Thermodynamic cooperativity of cosubstrate binding and cation selectivity of Salmonella typhimurium MelB

The Na⁺-coupled melibiose symporter MelB, which can also be coupled to H⁺ or Li⁺ transport, is a prototype for the glycoside-pentoside-hexuronide:cation symporter family. Although the 3-D x-ray crystal structure of Salmonella typhimurium MelB (MelB_St) has been determined, the symport mechanisms for the obligatory coupled transport are not well understood. Here, we apply isothermal titration calorimetry to determine the energetics of Na⁺ and melibiose binding to MelB_St, as well as protonation of this transporter. Studies of the thermodynamic cycle for the formation of the Na⁺-MelB_St-melibiose ternary complex at pH 7.45 reveal that the binding of Na⁺ and melibiose is cooperative. The binding affinity for one substrate (Na⁺ or melibiose) is increased by the presence of the other by about eightfold. The coupling free energies (ΔΔG) of either substrate binding are ∼5 kJ/mol, and binding of both substrates releases a free energy of ∼35 kJ/mol. Measurements of the Na⁺-binding enthalpy at three different pH values, including the pK_a value of MelB, indicate that the binding of one Na⁺ displaces one H⁺ per MelB_St molecule. In addition, the absolute dissociation constants for Na⁺ and H⁺, determined by competitive binding, show that MelB_St is selective for H⁺ over Na⁺ by ∼1,000-fold at a pK_a of 6.25. Thus, the Na⁺ coupling in MelB_St is based not on ion selectivity but on ion concentrations and competitive binding because of a much higher Na⁺ concentration under physiological conditions. Such a selectivity feature seems to be common for membrane transport proteins that can bind both H⁺ and Na⁺ at a common site.