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Abstract Energy-coupling factor (ECF)-type transporters mediate the uptake of micronutrients in many bacteria. They consist of a substrate-translocating subunit (S-component) and an ATP-hydrolysing motor (ECF module) Previous data indicate that the S-component topples within the membrane to alternately expose the binding site to either side of the membrane. In many ECF transporters, the substrate-free S-component can be expelled from the ECF module. Here we study this enigmatic expulsion step by cryogenic electron microscopy and reveal that ATP induces a concave-to-convex shape change of two long helices in the motor, thereby destroying the S-component’s docking site and allowing for its dissociation. We show that adaptation of the membrane morphology to the conformational state of the motor may favour expulsion of the substrate-free S-component when ATP is bound and docking of the substrate-loaded S-component after hydrolysis. Our work provides a picture of bilayer-assisted chemo-mechanical coupling in the transport cycle of ECF transporters.

Energy coupling factor (ECF) transporters are responsible for the uptake of micronutrients and consist of an integral membrane unit, the S-component, which confers substrate specificity. Here, authors present multi-scale molecular dynamics simulations and in vitro experiments to study the molecular toppling mechanism of the S-component of a folate-specific ECF transporter.