Membrane mediated toppling mechanism of the folate energy coupling factor transporter

Abstract

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.

Fig. 1. ECF-FolT2 complex structure and transport cycle.

a The S-component in its folate-bound conformation, b the folate-bound S-component binds to the ECF module (consisting of the EcfA, EcfA′, and EcfT subunits) in two possible orientations, canonical (upper part), or toppled (lower part). The orientation of the S-component is represented by the yellow line. The brown arrow shows the direction of the bilayer normal, c the S-component binds the ECF module to form the ECF complex. The S-component is in green, EcfT is in red, and EcfA/EcfA′ is in white surface. The bilayer is shown schematically.

Fig. 2. Orientation of the S-component.

a Snapshots of the S-component in canonical and toppled orientations. The S-component is shown in green and helix 5 in yellow. Both are plotted in van der Waals CG surface representation. The phosphate groups and tails of the lipids are shown in orange and cyan, respectively, with magenta for unsaturated parts of the tails. b Bar graph of orientational preference of the S-component starting from toppled state in different lipid environments in either apo or holo form (a: POPE-POPG-CL-holo S-component (ratio: 70:25:5), b: POPE-POPG-CL-apo S-component (ratio: 70:25:5), c: POPE-POPG-POPC-holo S-component (ratio: 60:20:20), d: DOPE-DOPG-DOPC-holo S-component (ratio: 60:20:20), e: DOPE-DOPG-DOPC-holo S-component (ratio: 50:13:37), f: DOPE-DOPG-DOPC-holo S-component (ratio: 30:30:40), g: DPPC-holo S-component, h: POPE-POPG-CL-holo RibU S-component (ratio: 70:25:5)). The error bars represent standard deviation. The lipid abbreviations are according to Table 1. Source data are provided as a Source Data file.

Fig. 3. ECF-FolT2 induced membrane deformation.

a MD snapshot showing the local membrane bending around the ECF module. EcfT is colored in red, EcfA and EcfA′ in white, and the phosphate groups of the lipids are colored in orange. Lipid tails are depicted in cyan with magenta for the unsaturated parts. b Curvature map around the ECF module (outline) for the upper leaflet, Lx and Ly denote bilayer lateral directions. c Membrane thickness map around the ECF module.

Fig. 4. Dissociation of S-component from ECF complex.

Potential of mean force of ECF module and S-component association (blue). Error bars were computed with bootstrapping. Average tilt angle between helix 5 and the positive z-direction of the simulation box along the dissociation pathway (red). Error bars are standard deviations. Representative snapshots of ECF and S-component along the dissociation pathway are shown. Toppled state is on the left and canonical state is on the right. Color coding is the same as Fig. 1. Helix 5 is highlighted in yellow. Source data are provided as a Source Data file.

Fig. 5. Folate uptake activity depends on lipid composition.

Folate uptake by ECF-FolT2 reconstituted in proteoliposomes was measured with liposomes made of different phospholipids and normalized to activity observed in E. coli polar lipids mixed with egg yolk PC (3:1 w/w) (E. coli PC). a Effect of tail composition with similar headgroup distribution. DO refers to only DO lipids, and PO to only PO lipids. b Effect of headgroup (PE) composition with same tail composition (DO). PE54, PE27, and PE00, contain 54%, 27%, and 0% PE, respectively. ADP indicated a negative control in which proteoliposomes with E. coli PC lipid composition were loaded with ADP instead of ATP. Exact lipid compositions can be found in Table 2. Error bars are standard deviations. Source data are provided as a Source Data file.

Fig. 6. Mechanism proposed for the transport of folate by the group II ECF transporter ECF-FolT2.

Once the open (canonical) S-component binds folate there are two pathways that the substrate-bound S-component can follow to bind the hydrophobic surface of the ECF module. In the first, the S-component can topple over inside the membrane possibly due to local fluctuations of the membrane and bind the ECF module in the orientation observed in crystallographic structures of ECF complexes. As a second pathway, the S-component can slide towards the ECF module thanks to the negative bending around the ECF module.