ATP-binding cassette (ABC) transporters are molecular machines that are involved in a wide range of physiological processes, including antigen presentation by TAP, a key player in adaptive immunity. TAP and its bacterial homolog TmrAB use ATP to translocate peptides across membranes. To elucidate the mechanism of substrate transport, we employed a single-molecule FRET sensor to visualize single-translocation events by individual ABC transporters, overcoming the limitations of ensemble averaging. Using this approach, we tracked the membrane translocation of single peptides driven by a conformational switch from the inward- to the outward-facing state. In a slow-turnover TmrAB variant, even in the absence of Mg2+, we show that ATP binding alone is sufficient to drive peptide translocation. Structural analyses via cryogenic electron microscopy reveal that ATP binding induces an outward-facing conformation in both wild-type and mutant TmrAB, even without Mg2+. In wild-type TmrAB, this was sufficient for a single peptide translocation. However, Mg2+ was essential for the later stages of ATP hydrolysis and transporter resetting. Together, these results reveal a direct mechanistic link between ATP binding and substrate translocation at single-molecule resolution, providing new insights into the catalytic cycle of ABC transporters.

Supplementary material and Source data contributing to the paper 'Single-molecule dynamics reveals ATP binding alone powers substrate translocation by an ABC transporter'.