In vitro reconstitution of dynamically interacting integral membrane subunits of energy-coupling factor transporters

Abstract

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.

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

“…The inward-facing apo state is a structurally stable conformation ECF-FolT2 in lipid nanodiscs adopts an inward-facing apo conformation, where the S-component FolT2 assumes a substrate-free 'toppled' state with both nucleotide-binding proteins in the open and nucleotide-free conformation. This conformation resembles the post-substrate-release state (3,4,(6)(7)(8)(9) obtained in previous crystallographic studies on ECF transporter complexes (Fig. 1A), showing that the inward-facing apo conformation represents a structurally stable conformation in a near native and non-native environments.…”

“…The modular nature of ECF transporter complexes requires a degree of flexibility to facilitate the docking and toppling of the S-components. The differences in the interactions observed between EcfT and the S-component are in agreement with the dynamic nature of the transmembrane region of EcfT (3,4,6,9), which is an important feature to accommodate different S-components and to facilitate the toppling of the substrate-bound S-component during the transport cycle (3)(4)(5)(6). Although unrelated in sequence and in substrate specificities, different S-components have a common structural fold and can dock onto the same ECF module (3,4,6,20,21).…”

“…The former three proteins form a tripartite module of ~ 93 kDa (named the energising or ECF module), while the S-component of ~ 19 kDa can be either associated with the ECF module ("full complex"), or be present in the membrane as a solitary protein. ECF transporter complexes catalyse the uptake of micronutrients such as vitamins and transition metals in bacteria and their substrate specificity is mediated by a different S-component for each substrate (2,3). X-ray crystal structures suggest that the S-component must topple within the membrane to bring the substrate from the outside to the inside of the cell (3)(4)(5).…”

“…ECF transporter complexes catalyse the uptake of micronutrients such as vitamins and transition metals in bacteria and their substrate specificity is mediated by a different S-component for each substrate (2,3). X-ray crystal structures suggest that the S-component must topple within the membrane to bring the substrate from the outside to the inside of the cell (3)(4)(5). Coarse-grained molecular dynamics simulations have indicated that the membrane bends and thins near the ECF module, providing a favourable environment for the S-component to topple within the lipid bilayer (5).…”

“…However, experimental evidence for membrane deformations around ECF transporter complexes, required to substantiate this idea, is lacking. Most X-ray crystallisation methods use detergent-solubilised proteins, rendering a direct visualising of the bilayer environment, and consequently the correct positioning of the protein bilayer, impossible (3,4,(6)(7)(8)(9). Therefore, structures of ECF transporter complexes embedded in a more native environment are required to understand their interplay with the lipid bilayer.…”