BPS2026 – Selective elimination of Na+ binding in Klebsiella pneumoniae MelB

Salmonella enterica serovar Typhimurium melibiose permease (MelB St ), a member of the major facilitator superfamily of transporters, mediates cotransport of melibiose with H + , Na + , or Li + . It was reported that the Klebsiella pneumoniae ortholog MelB Kp lost cotransport with Na + but increased H + -coupled melibiose transport. Here, binding measurements using right-side-out membrane vesicles with Trp→dansyl galactoside FRET assay showed that MelB Kp did not bind Na + but could bind Li + . Five non-conserved positions, three at helix II and two at loop 1-2 , were mutated to the sidechains in MelB St alone or in combination. Mutant A58N within the cation-binding pocket, with all necessary components for Na + binding, showed poor melibiose/Na + or Li + symport activities while regaining measurable affinities for both cations. Adding the mutation L54W significantly increased all three modes of transport, especially with Na + and Li + , and their binding affinities. Further mutations at the other three positions decreased all three modes of transport with little effect on Na + or Li + binding. Notably, mutant L54W did not bind Na + or catalyze the Na + /melibiose co-transport, but bound Li + and carried out significant Li + -coupled transport. All mutations slightly inhibited melibiose-binding affinity and exhibited a similar level of membrane expression, except for the less-expressed L54W/A58N mutant. Our results support two major conclusions: (1) Asn at position 58 is necessary for Na + recognition but not essential for Li + binding, which confirms the previous finding that Li + can bind to a subset of the Na + -binding pocket; (2) the hydrophobic Ala in the cation pocket of MelB Kp enhances H + -coupled melibiose transport, likely by raising the p K a of Asp59. This study has contributed to our understanding of the mechanisms behind cation selectivity. Interestingly, MelB Kp succeeded in evolving the cation selectivity by selectively eliminating Na + recognition and promoting H + -coupled transport activity.