Crystal Structures of a Melibiose Symporter MelB with Bound Sugar Substrate

Abstract

MelB is a well-studied symporter from the major facilitator superfamily of transporters, and its mammalian homologues play important roles in the delivery of essential omega-3 fatty acids into brain and eyes. The bacterial MelB is selective for galactose or galactosides, and catalyzes the obligatory cotransport of the sugar with Na+, Li+, or H+. Previous 3-D crystal structure of the MelBSt of Salmonella thyphimurium shows that MelB adopts a typical MFS-fold, and the thermodynamic ligand binding studies reveal that cooperative binding of Na+ and melibiose is the core mechanism for the obligatory symport. To gain insights into the substrate binding, we solved three sugar-bound crystal structures from two MelBSt mutants that are compromised on Na+ binding. The structures with bound nitrophenyl-α-D-galactoside or melibiose-based detergent dodecyl-β-d-melibioside (DDMB) were refined to a resolution between 3.05 - 3.25 Å. All three structures are nearly identical at an outward-facing conformation; each one contains a á-galactoside molecule in the middle of protein. The binding interactions of galactosyl moiety from each ligand are indistinguishable, revealing a selection pocket, and the non-galactosyl moieties occupy differently. The active site displays a strong salt-bridge/hydrogen-bonding network that provides multiple polar interactions to each of four OH groups on the galactosyl moiety, and the galactosyl ring is sandwiched by two aromatic sidechains via aromatic stacking interactions. There is little or no interaction toward the non-galactosyl moieties, and the entire 12-carbon tail of DDMB lies within the internal cavity. The localized interactions towards the specific galactosyl moiety and the large internal cavity allow MelB to accommodate a variety of galactosides with different structures. The proposed Na+-binding pocket with no bound Na+ seems to lie in a close proximity to the bound sugar. These structures represent a Na+-free, galactoside-bound binary complex of MelB.