Molecular Basis for the Cation Selectivity of Salmonella typhimurium Melibiose Permease

Abstract

Cation selectivity and coupling are important attributes of cation-coupled symporters. Salmonella typhimurium melibiose permease (MelB_St) catalyzes the co-transport of galactosides with cations (H⁺, Li⁺, or Na⁺). 3-D crystal structures of MelB_St have revealed the molecular recognition for sugar substrates, but the cation binding and coupling mechanisms have not been defined to atomic levels. In its human homolog MFSD2A, a lethal mutation was mapped at its Na⁺-binding pocket; however, none of the structures in this subfamily resolved its cation binding. In this study, molecular dynamics simulations reveal the binding interactions of Na⁺ and Li⁺ with MelB_St. Interestingly, Thr121, the lethal mutation position in MFSD2A, forms stable interaction with Na⁺ but is at a distance from Li⁺. Most mutations among 11 single-site Thr121 mutants of MelB_St exhibited little effects on the galactoside binding, but largely altered the cation selectivity with severe inhibitions on Na⁺ binding. Few mutants (Pro and Ala) completely lost the Na⁺ binding and Na⁺-coupled transport, but their Li⁺ or H⁺ modes of activity were largely retained. It can be concluded that Thr121 is necessary for Na⁺ binding, but not required for the binding of H⁺ or Li⁺, so a subset of the Na⁺-binding pocket is enough for Li⁺ binding. In addition, the protein stability for some mutants can be only retained in the presence of Li⁺, but not by Na⁺ due to the lack of affinity. This finding, together with other identified thermostable mutants, supports that the charge balance of the cation-binding site plays an important role in MelB_St protein stability.

Keywords: 3-D structure; MD simulations; cation-coupled symporter; ligand binding; membrane protein stability; structure and function analysis.