Insights into the bilayer-mediated toppling mechanism of a folate-specific ECF transporter by cryo-EM

Abstract

Energy-coupling factor (ECF)–type transporters are small, asymmetric membrane protein complexes (∼115 kDa) that consist of a membrane-embedded, substrate-binding protein (S component) and a tripartite ATP-hydrolyzing module (ECF module). They import micronutrients into bacterial cells and have been proposed to use a highly unusual transport mechanism, in which the substrate is dragged across the membrane by a toppling motion of the S component. However, it remains unclear how the lipid bilayer could accommodate such a movement. Here, we used cryogenic electron microscopy at 200 kV to determine structures of a folate-specific ECF transporter in lipid nanodiscs and detergent micelles at 2.7- and 3.4-Å resolution, respectively. The structures reveal an irregularly shaped bilayer environment around the membrane-embedded complex and suggest that toppling of the S component is facilitated by protein-induced membrane deformations. In this way, structural remodeling of the lipid bilayer environment is exploited to guide the transport process.

Distinct energy-coupling factor transporter subunits enable flavin acquisition and extracytosolic trafficking for extracellular electron transfer inListeria monocytogenes

“…These studies reveal that ECF-S remains monomeric in the absence of substrate, likely adopting an ‘outward’ facing orientation that enables substrate binding from the extracytosolic space. 22,34,35 Once substrate is bound, ECF-S undergoes a ‘toppling’ conformational change to an ‘inward’ orientation where it engages the ECF-T, ECF-A, ECF-A’ complex in a manner that facilitates substrate release into the cytosol. ATP hydrolysis in the ECF complex then causes release of apo ECF-S, which reverts to its monomeric ‘outward’ orientation ( Figure 5A ).…”

“…ATP hydrolysis in the ECF complex then causes release of apo ECF-S, which reverts to its monomeric ‘outward’ orientation ( Figure 5A ). 22,35 Considering the similarity of subunits, the proposed mechanism of ECF import has implications for how EetB might function in FAD export. Simply reversing the relationship between ECF-S with the rest of the ECF transporter complex could reverse the direction of transport ( Figure 5B ).…”

Distinct Energy-Coupling Factor Transporter Subunits Enable Flavin Acquisition and Extracytosolic Trafficking for Extracellular Electron Transfer in Listeria monocytogenes

“…While little is known about the mechanism of ECF export, previous research has generated considerable evidence about the basis of ECF import. These studies reveal that ECF-S remains monomeric in the absence of a substrate, likely adopting an “outward”-facing orientation that enables substrate binding from the extracytosolic space ( 23 , 35 , 36 ). Once the substrate is bound, ECF-S undergoes a “toppling” conformational change to an “inward” orientation where it engages the ECF-T–ECF-A–ECF-A′ complex in a manner that facilitates substrate release into the cytosol.…”

“…ATP hydrolysis in the ECF complex then causes the release of apo-ECF-S, which reverts to its monomeric outward orientation ( Fig. 5A ) ( 23 , 36 ). Considering the similarity of subunits, the proposed mechanism of ECF import has implications for how EetB might function in FAD export.…”

Hit optimization by dynamic combinatorial chemistry on Streptococcus pneumoniae energy-coupling factor transporter ECF-PanT

“…Molecular dynamics (MD) simulations and cryo-EM spectroscopy revealed that the protein-induced membrane deformations enable the toppling of the S-component across the membrane and its interaction with the ECF module. 4,5 This unique architecture and mechanism of action make the ECF transporters fascinating and challenging targets. Over the past decade, several structural and functional studies have unravelled their mechanism of transport.…”