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

TLDR: 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.

Abstract: 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 (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 pKa 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 pKa 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.