Single-molecule visualization of ATP-induced dynamics of the subunit composition of an ECF transporter complex under turnover conditions

Nat Commun. 2025 May 13;16(1):4448. doi: 10.1038/s41467-025-59674-6.

Abstract

Energy-Coupling Factor (ECF) transporters are ATP-binding cassette (ABC) transporters essential for uptake of vitamins and cofactors in prokaryotes. They have been linked to pathogen virulence and are potential targets for antimicrobials. ECF transporters have been proposed to use a unique transport mechanism where a substrate-translocating subunit (S-component) dynamically associates with and dissociates from an ATP-hydrolyzing motor (ECF module). This model is contentious, because it is based largely on experimental conditions without compartments or continuous bilayers. Here, we used single-molecule spectroscopy to investigate the conformational dynamics of the vitamin B12 transporter ECF-CbrT in membranes under vectorial transport conditions. We observed ATP hydrolysis-dependent dissociation of the S-component CbrT from, and re-association with the ECF module, in absence and presence of vitamin B12 consistent with futile ATP hydrolysis activity. The single-molecule spectroscopy experiments suggest that S-component expulsion from and re-association with the ECF module are an integral part of the translocation mechanism.

MeSH terms

  • ATP-Binding Cassette Transporters* / chemistry
  • ATP-Binding Cassette Transporters* / genetics
  • ATP-Binding Cassette Transporters* / metabolism
  • Adenosine Triphosphate* / metabolism
  • Biological Transport
  • Escherichia coli / metabolism
  • Escherichia coli Proteins* / chemistry
  • Escherichia coli Proteins* / metabolism
  • Hydrolysis
  • Protein Conformation
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism
  • Single Molecule Imaging* / methods
  • Vitamin B 12 / metabolism

Substances

  • Adenosine Triphosphate
  • ATP-Binding Cassette Transporters
  • Vitamin B 12
  • Escherichia coli Proteins
  • Protein Subunits