In vivo and in vitro characterizations of melibiose permease (MelB) conformation-dependent nanobodies reveal sugar-binding mechanisms

Salmonella enterica serovar Typhimurium melibiose permease (MelB_St) is a prototype of the Na⁺-coupled major facilitator superfamily transporters, which are important for the cellular uptake of molecules including sugars and small drugs. Although the symport mechanisms have been well-studied, mechanisms of substrate binding and translocation remain enigmatic. We have previously determined the sugar-binding site of outward-facing MelB_St by crystallography. To obtain other key kinetic states, here we raised camelid single-domain nanobodies (Nbs) and carried out a screening against the WT MelB_St under 4 ligand conditions. We applied an in vivo cAMP-dependent two-hybrid assay to detect interactions of Nbs with MelB_St and melibiose transport assays to determine the effects on MelB_St functions. We found that all selected Nbs showed partial to complete inhibitions of MelB_St transport activities, confirming their intracellular interactions. A group of Nbs (714, 725, and 733) was purified, and isothermal titration calorimetry measurements showed that their binding affinities were significantly inhibited by the substrate melibiose. When titrating melibiose to the MelB_St/Nb complexes, Nb also inhibited the sugar-binding. However, the Nb733/MelB_St complex retained binding to the coupling cation Na⁺ and also to the regulatory enzyme EIIA^Glc of the glucose-specific phosphoenolpyruvate/sugar phosphotransferase system. Further, EIIA^Glc/MelB_St complex also retained binding to Nb733 and formed a stable supercomplex. All data indicated that MelB_St trapped by Nbs retained its physiological functions and the trapped conformation is similar to that bound by the physiological regulator EIIA^Glc. Therefore, these conformational Nbs can be useful tools for further structural, functional, and conformational analyses.