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

## TL;DR

This study uses single-molecule techniques to visualize how an ECF transporter complex changes during vitamin B12 transport, revealing dynamic subunit interactions.

## Contribution

The study provides direct evidence for dynamic S-component association/dissociation in ECF transporters under physiological conditions.

## Key findings

- ATP hydrolysis drives CbrT dissociation and re-association with the ECF module.
- Dynamics occur both with and without vitamin B12, indicating futile ATP hydrolysis.
- Single-molecule FRET visualizes the transport mechanism in real time in membranes.

## 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.

The association and dissociation dynamics of the ECF transporter complex for vitamin B12 are visualized by single-molecule FRET, highlighting the original transport mechanism of this group of ABC transporters.