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

ABSTRACT: 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

SUBMITTER: Katsube S 

PROVIDER: S-EPMC10374971 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature