{"doi":"10.7554/elife.92462.1","title":"Mimicking the regulatory state of a major facilitator superfamily sugar transporter","abstract":"Abstract While many 3D structures of cation-coupled transporters have been determined, the mechanistic details governing the obligatory coupling and functional regulations still remain elusive. The bacterial melibiose transporter (MelB) is a prototype of the Na+-coupled major facilitator superfamily transporters. With a conformational nanobody (Nb), we determined an inward-facing Na+-bound, low-sugar affinity cryoEM structure. It is the first structure of a major facilitator superfamily (MFS) transporter with experimentally determined cation binding, and also a structure mimicking the physiological regulatory state of MelB under the global regulator EIIAGlc of the glucose-specific phosphoenolpyruvate:phosphotransferase system. Collectively with the available outward-facing sugar-bound structures and a large body of functional analysis, we identified that only inner barrier that exists in the outward-facing conformation contributes to the sugar selectivity pocket. When the inner barrier is broken as shown in the inward-facing conformation, the sugar selectivity pocket is also broken, resulting in a decreased sugar-binding affinity by greater than 30-fold, which can facilitate the substrate release and accumulation intracellularly. The inner/outer barrier shifting directly regulates the sugar-binding affinity, with no effect on the cation binding as also suggested by molecular dynamics simulations. Furthermore, the use of the inward-facing conformation-specific Nb in combination with the hydron/deuterium exchange mass spectrometry allowed us to identify dynamic regions linked to the inner barrier-specific charged network and critical for the barrier switching mechanisms. The complementary results provided structural, dynamic, and regulatory insights into the mobile barrier mechanism for cation-coupled symport.","journal":null,"year":2023,"id":413908,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9362,"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":457007,"name":"Yuqi Shi","orcid":"0009-0000-0825-7933","position":1,"is_corresponding":false},{"id":722130,"name":"Satoshi Katsube","orcid":"0000-0002-3312-7476","position":2,"is_corresponding":false},{"id":1085269,"name":"Katleen Willibal","orcid":null,"position":3,"is_corresponding":false},{"id":1084749,"name":"Nathan D. Burrows","orcid":"0000-0002-3973-1017","position":4,"is_corresponding":false},{"id":286104,"name":"Patrick Mitchell","orcid":"0000-0002-3458-6930","position":5,"is_corresponding":false},{"id":1084750,"name":"Amirhossein Bakhtiiari","orcid":"0000-0002-3979-0973","position":6,"is_corresponding":false},{"id":1085270,"name":"Samantha Stanfield","orcid":null,"position":7,"is_corresponding":false},{"id":263120,"name":"Els Pardon","orcid":"0000-0002-2466-0172","position":8,"is_corresponding":false},{"id":519647,"name":"H. Ronald Kaback","orcid":"0000-0003-0683-5810","position":9,"is_corresponding":false},{"id":630200,"name":"Ruibin Liang","orcid":"0000-0001-8741-1520","position":10,"is_corresponding":false},{"id":263123,"name":"Jan Steyaert","orcid":"0000-0002-3825-874X","position":11,"is_corresponding":false},{"id":89978,"name":"Rosa Viner","orcid":"0000-0003-0550-5545","position":12,"is_corresponding":false},{"id":563736,"name":"Lan Guan","orcid":"0000-0002-2274-361X","position":13,"is_corresponding":false},{"id":587009,"name":"Parameswaran Hariharan","orcid":"0000-0002-6020-1547","position":0,"is_corresponding":true}],"reference_count":82,"raw_metadata":null,"created_at":"2026-07-19T01:22:01.321790Z","pmid":null,"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":[]}