Insights into the Cation Selectivity and Cooperativity with Sugar in Salmonella Typhimurium Melibiose Permease

Abstract

The melibiose permease of Salmonella typhimurium (MelBSt), a member of Major Facilitator Superfamily, utilizes the electrochemical gradient of Na+, Li+ or H+ for accumulation of galactosides. Based on crystal structures and functional studies, it has been proposed that MelBSt has a single galactoside-binding site and all three cations compete for a single cation site. Using purified wild-type or mutants MelBSt, direct measurement of Na+ or melibiose binding were performed with isothermal titration calorimetry. The results show that the binding affinity for Na+ or melibiose is mutually increased by about 6-8 fold, exhibiting a positive cooperativity. To reveal the cation selectivity between Na+ and H+, the dissociation constant (Kd) of Na+ binding was measured as a function of pH. The absolute KD value for Na+ of 600µM and for H+of 0.4 µM (i.e. pKa = 6.4) are determined, showing that MelBSt selects for H+ over Na+. With a pKa value slightly lower than the neutral pH, MelB is able to use either H+ or Na+ as the coupling cation. Furthermore, H+ release during Na+ binding was analyzed by measuring the overall heat changes in three reaction buffers with different ionization enthalpies. While Kd for Na+ is similar, the enthalpy changes obtained in the three buffers fit to a linear function with a slope of −0.3 and −0.03 at pH 7.45 or 8.2, respectively. It is likely that about 30% MelBSt proteins engage H+ release during Na+ binding at pH 7.45 and nearly all MelBSt is deprotonated at pH 8.2. The results also support the notion that Na+ and H+ exclusively bind to a single site. This study provides insights into the molecular mechanisms operated by MelB to utilize either Na+ or H+.