Crystal structure of a folate energy-coupling factor transporter from Lactobacillus brevis

Abstract

ATP-binding cassette (ABC) transporters, composed of importers and exporters, form one of the biggest protein superfamilies that transport a variety of substrates across the membrane, powered by ATP hydrolysis. Most ABC transporters are composed of two transmembrane domains and two cytoplasmic nucleotide-binding domains. Also, importers from prokaryotes usually have extra solute-binding proteins in the periplasm that are responsible for the binding of substrates. Structures of importers have been reported that suggested a two-state model for the transport mechanism. Energy-coupling factor (ECF) transporters belong to a new class of ATP-binding cassette importers. Each ECF transporter comprises an energy-coupling module consisting of a transmembrane T protein (EcfT), two nucleotide-binding proteins (EcfA and EcfA'), and another transmembrane substrate-specific binding S protein (EcfS). Despite the similarities with ABC transporters, ECF transporters have different organizational and functional properties. The lack of solute-binding proteins in ECF transporters differentiates them clearly from the canonical ABC importers. Previously reported structures of the EcfS proteins RibU and ThiT clearly demonstrated the binding site of substrate riboflavin and thiamine, respectively. However, the organization of the four different components and the transport mechanism of ECF transporters remain unknown. Here we present the structure of an intact folate ECF transporter from Lactobacillus brevis at a resolution of 3 Å. This structure was captured in an inward-facing, nucleotide-free conformation with no bound substrate. The folate-binding protein FolT is nearly parallel to the membrane and is bound almost entirely by EcfT, which adopts an L shape and connects to EcfA and EcfA' through two coupling helices. Two conserved XRX motifs from the coupling helices of EcfT have a vital role in energy coupling by docking into EcfA-EcfA'. We propose a transport model that involves a substantial conformational change of FolT.

Structural and mechanistic insights into prokaryotic energy-coupling factor transporters

“…Both of the L. brevis ECF transporters that were recently crystallized belong to group II and consist of the same ECF module but contain different S components 21,22 . One complex (ECF-FolT) contains FolT, which is the S component that transports folates 21 ; and the other complex (ECF-HmpT) contains thiamine precursor transporter HmpT 22 , which is the S component that was originally predicted to transport hydroxy methyl pyrimidine 6 .…”

“…One complex (ECF-FolT) contains FolT, which is the S component that transports folates 21 ; and the other complex (ECF-HmpT) contains thiamine precursor transporter HmpT 22 , which is the S component that was originally predicted to transport hydroxy methyl pyrimidine 6 . HmpT has been renamed PdxU2 as genomic context analysis suggests that it transports pyridoxin (The SEED database 27 ).…”

“…Similarly to other membrane transport proteins, delineating the mechanistic details of transport requires high-resolution crystal structures and the structures of two Lactobacillus brevis ECF transporters have recently been obtained 21,22 . In this Progress article, I describe the genetic organization, overall stoichiometry and structural composition of ECF transporters and discuss a novel mechanism of transport on the basis of the recent structural insights.…”

“…As different S components lack a high degree of sequence similarity, the use of the same ECF module by group II transporters raises an intriguing question: how does the same ECF module recognize unrelated S components? The recent crystal structures 21,22 provide some clues to explain how this might be possible (see below).…”

“…1c). The mechanism of transport that is used by the solitary BioY proteins is unknown, but the structures of the complete ECF complexes 21,22 might provide some clues (see below). It has also been reported that the S components of two different Rhodobacter capsulatus group I transporters (BioY and the bipartite S component for cobalt CbiMN) are able to mediate transport in the absence of their respective ECF modules (BioMN and CbiQO, respectively) 8 .…”