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 (MelBSt ) 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 MelBSt , as well as protonation of this transporter. Studies of the thermodynamic cycle for the formation of the Na+ -MelBSt -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 (ΔΔ G ) 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 p K a value of MelB, indicate that the binding of one Na+ displaces one H+ per MelBSt molecule. In addition, the absolute dissociation constants for Na+ and H+ , determined by competitive binding, show that MelBSt is selective for H+ over Na+ by ∼1,000-fold at a p K a of 6.25. Thus, the Na+ coupling in MelBSt 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.