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.

Keywords: ABC transporter; cryo-EM; lipid bilayer deformation; membrane transport.

Fig. 1.

Cryo-EM structure of the folate-specific ECF transporter complex in the inward-facing apo conformation embedded in lipid nanodiscs. (A) Atomic model of ECF-FolT2 in lipid nanodiscs as viewed from the membrane plane shown in cartoon representation built into the cryo-EM density map (transparent contours at 9.5 σ) with an overlay of the density corresponding to the lipid nanodisc drawn transparent (unsharpened map lowpass filtered to 6 Å and contoured at 0.325 σ). The two nucleotide-binding proteins (EcfA in red and EcfA’ in orange) and the two membrane-embedded proteins (EcfT in blue and FolT2 in yellow) form together ECF-FolT2. Water molecules shown as green spheres. Approximate boundaries of the membrane are indicated by black dotted lines. (B–E) Interactions of the S component with EcfT and EcfA’. Interactions formed between FolT2 to EcfT in the cryo-EM structure (B) and in the AMP-PNP–bound X-ray crystal structure of ECF-FolT2 (PDB: 5d3m) (C). Interactions formed between FolT2 to EcfA’ in the cryo-EM structure (D) and in the AMP-PNP–bound structure (E). Individual polypeptides are colored as in (A). Highlighted residues are represented in the ball-and-stick style. Hydrogen bonds and π-interactions are shown as black and green dotted lines. Selected structural elements and residues are labels. Schematic representations Above panels (B and D) indicate the zoomed in regions for panels Below.

Fig. 2.

Membrane deformations mediated by ECF-FolT2. Densities for cryo-EM maps displayed as described in Fig. 1. (A) Viewed from the membrane plane on the side of the exit pathway for folate from the cavity in FolT2. The exit pathway is indicated by a green arrow. The pivot point of the membrane and coupling domains of EcfT at conserved residue Pro71 in THM3 is indicated by an orange sphere. (B) Viewed from the membrane plane as in (A) but rotated by 180° highlighting the deformation of the lipid nanodisc at the cytoplasmic base of FolT2. The approximate thickness of the lipid nanodiscs was determined using Chimera X (22) and indicated by black arrows. Selected structural elements are labeled. Approximate boundaries of the membrane are indicated by black dotted lines.

Fig. 3.

Protein–lipid interactions. (A) Lipids at the cytoplasmic base of FolT2. The Inset is a zoomed view of the large boxed panel rotated by 90°, highlighting the density (blue) for another acyl chain (labeled I*), which either belongs to a second lipid molecule in this region or forms with the remaining density of lipid I (purple density) a cardiolipin molecule. (B) Lipids near the exit pathway for the substrate. (C) Lipids around EcfT viewed from the membrane plane. Global views of the interpreted lipids (green spheres) around the membrane components EcfT and FolT2 (surface representation) within the lipid nanodisc (contoured as in Fig. 2) as viewed from the membrane plane are shown next to schematic representations of the full complex. Large boxed panels represent zoomed views of regions as highlighted in the schematic representations. Proteins are shown in cartoon and surface representation. Modeled lipids are shown in stick representation (green) with the corresponding cryo-EM density contoured at 6.5 σ (purple). Roman numbers in the panel represent individual lipids or lipid groups referred to in the main text. Selected structural elements are labeled. The contours of the lipid nanodisc (unsharpened map lowpass filtered to 6 Å and contoured at 0.325 σ) are indicated in black. The exit pathway in (B) is indicated by a green arrow, and the inner and outer leaflet of the membrane are indicated by double-headed arrows (A–C). In all representations, proteins are colored according to Fig. 1.

Fig. 4.

Substrate transport by a bilayer-mediated toppling mechanism of ECF transporter complexes. Schematic representation of the different states of the transport cycle by ECF transporter complexes with exchangeable S components (so-called group II ECF transporters) (24). State 1: The substrate-bound S component docks to a freely available ECF module, which is in a slightly tiled conformation and thins and bends the membrane. The S component topples within the membrane. State 2: The ECF transporter complex assumes the inward-facing apo conformation, in which the bound substrate is released into the cell. The S component locally deforms the membrane by which the positively charged base of the FolT2 remains solvent exposed. State 3: Binding and hydrolysis of Mg-ATP results in the rotation of the apo S component to the outward-facing conformation and the release from the ECF module for renewed substrate binding. The ECF module tilts and bends the membrane for binding a substrate-bound S component. State 3 is based on structural evidence by cryo-EM (this work) and X-ray crystallographic studies. The remaining states are based on molecular dynamics simulations (5).