Energy-coupling factor (ECF)–type ATP-binding cassette (ABC) transporters catalyze membrane transport of micronutrients in prokaryotes. Crystal structures and biochemical characterization have revealed that ECF transporters are mechanistically distinct from other ABC transport systems. Notably, ECF transporters make use of small integral membrane subunits (S-components) that are predicted to topple over in the membrane when carrying the bound substrate from the extracellular side of the bilayer to the cytosol. Here, we review the phylogenetic diversity of ECF transporters as well as recent structural and biochemical advancements that have led to the postulation of conceptually different mechanistic models. These models can be described as power stroke and thermal ratchet. Structural data indicate that the lipid composition and bilayer structure are likely to have great impact on the transport function. We argue that study of ECF transporters could lead to generic insight into membrane protein structure, dynamics, and interaction.

Energy coupling factor (ECF) transporters are responsible for the uptake of micronutrients in bacteria and archaea. They consist of an integral membrane unit, the S-component, and a tripartite ECF module. It has been proposed that the S-component mediates the substrate transport by toppling over in the membrane when docking onto an ECF module. Here, we 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. Simulations reveal a strong bending of the membrane around the ECF module that provides a driving force for toppling of the S-component. The stability of the toppled state depends on the presence of non-bilayer forming lipids, as confirmed by folate transport activity measurements. Together, our data provide evidence for a lipid-dependent toppling-based mechanism for the folate-specific ECF transporter, a mechanism that might apply to other ECF transporters.

The energy-coupling factor (ECF) transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. Their central role in the metabolism of bacteria and absence in humans make the ECF transporters a potential antibacterial target, which can be further investigated making use of a selective chemical probe. Here, we report on the virtual screening, design, synthesis, structure–activity relationships (SARs) and coarse-grained molecular dynamics simulations of the first class of inhibitors of the ECF transporters. We investigated the mechanism of action of this chemical class and profiled the best hit compounds regarding their pharmaceutical properties. The optimized hit has a minimum inhibitory concentration (MIC) value of 2 µg/mL against Streptococcus pneumoniae, which opens up the possibility to use this chemical class to investigate the role of the ECF transporters in health and disease.

Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstituted system, required to substantiate this idea, is lacking. Here, we co-reconstituted ECF transporters for folate (ECF-FolT2) and pantothenate (ECF-PanT) into proteoliposomes, and assayed for crosstalk during active transport. The kinetics of transport showed that exchange of S-components is part of the transport mechanism. Competition experiments suggest much slower substrate association with FolT2 than with PanT. Comparison of a crystal structure of ECF-PanT with previously determined structures of ECF-FolT2 revealed larger conformational changes upon binding of folate than pantothenate, which could explain the kinetic differences. Our work shows that a minimal in vitro system with two reconstituted transporters recapitulates intricate kinetics behaviour observed in vivo.

Energy coupling factor (ECF) transporters take up micronutrients in Bacteria and Archaea. They consist of a membrane-embedded S-component that provides substrate specificity and a three-subunit ECF module that couples ATP hydrolysis to transport. The S-components ThiT (for thiamin) and NiaX (for niacin) from Lactococcus lactis form complexes with the same ECF module. Here, we assayed the uptake of thiamin and niacin in Escherichia coli cells expressing the transporter genes. We demonstrate that the two different S-components compete for the ECF module, and that competition is more efficient in the presence of the transported substrate. The data suggest that binding and release of the S-components is a step in the transport cycle.