{"doi":"10.1016/j.jbc.2023.104967","title":"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 (MFS) 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 four 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 super-complex. 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.","journal":"Journal of Biological Chemistry","year":2023,"id":360857,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":9,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9596,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1085269,"name":"Katleen Willibal","orcid":null,"position":1,"is_corresponding":false},{"id":1113089,"name":"Sangama Vemulapally","orcid":null,"position":2,"is_corresponding":false},{"id":587009,"name":"Parameswaran Hariharan","orcid":"0000-0002-6020-1547","position":3,"is_corresponding":false},{"id":1112691,"name":"Elena Tikhonova","orcid":"0000-0001-8252-6150","position":4,"is_corresponding":false},{"id":263120,"name":"Els Pardon","orcid":"0000-0002-2466-0172","position":5,"is_corresponding":false},{"id":519647,"name":"H. Ronald Kaback","orcid":"0000-0003-0683-5810","position":6,"is_corresponding":false},{"id":263123,"name":"Jan Steyaert","orcid":"0000-0002-3825-874X","position":7,"is_corresponding":false},{"id":563736,"name":"Lan Guan","orcid":"0000-0002-2274-361X","position":8,"is_corresponding":false},{"id":722130,"name":"Satoshi Katsube","orcid":"0000-0002-3312-7476","position":0,"is_corresponding":true}],"reference_count":44,"raw_metadata":null,"created_at":"2026-07-19T01:14:06.433417Z","pmid":"37380079","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}