Substrate Capture by ABC Transporters

Most ABC importers known to date employ a soluble substrate-binding protein to capture the ligand and donate the molecule to the translocator. The SBP can be a soluble periplasmic protein or tethered to the membrane via a lipid moiety or protein anchor or fused to the translocator. In the hybrid ABC transporters, multiple SBDs can be fused in tandem and provide several extracytoplasmic substrate-binding sites. A subset of ABC transporters employs a membrane-embedded S-component to capture the substrate. The S-component together with the ECF module also forms the translocation path for the substrate. Multiple S-components can associate consecutively with one and the same ECF module. An overview of the mechanism of substrate capture by different types of ABC transporters is presented, together with a scheme illustrating the alternating access mechanism for the overall transport process

Diversity of membrane transport proteins for vitamins in bacteria and archaea

BACKGROUND: All organisms use cofactors to extend the catalytic capacities of proteins. Many bacteria and archaea can synthesize cofactors from primary metabolites, but there are also prokaryotes that do not have the complete biosynthetic pathways for all essential cofactors. These organisms are dependent on the uptake of cofactors, or at least their precursors that cannot be synthesized, from the environment. Even in those organisms that contain complete biosynthetic pathways membrane transporters are usually present, because the synthesis of cofactors is more costly than uptake.SCOPE OF REVIEW: Here we give an overview of bacterial and archaeal transport systems for B-type vitamins, which are either cofactors or precursors thereof.MAJOR CONCLUSIONS: Prokaryotic vitamin transporters are extremely diverse, and found in many families of transporters. A few of these transport systems have been characterized in detail, but for most of them mechanistic insight is lacking.GENERAL SIGNIFICANCE: The lack of structural and functional understanding of bacterial vitamin transporters is unfortunate because they may be targets for new antibiotics. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins. Guest Editor: Bjorn Pedersen.</p

Substrate Capture by ABC Transporters

Most ABC importers known to date employ a soluble substrate-binding protein to capture the ligand and donate the molecule to the translocator. The SBP can be a soluble periplasmic protein or tethered to the membrane via a lipid moiety or protein anchor or fused to the translocator. In the hybrid ABC transporters, multiple SBDs can be fused in tandem and provide several extracytoplasmic substrate-binding sites. A subset of ABC transporters employs a membrane-embedded S-component to capture the substrate. The S-component together with the ECF module also forms the translocation path for the substrate. Multiple S-components can associate consecutively with one and the same ECF module. An overview of the mechanism of substrate capture by different types of ABC transporters is presented, together with a scheme illustrating the alternating access mechanism for the overall transport process.

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

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

Structural features of the glutamate transporter family

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate fi om the synaptic cleft and thus prevent neurotoxicity The proteins belong to a large and widespread family of secondary transporters, including bacterial glutamate, serine, and C-4-dicarboxylate transporters; mammalian neutral-amino-acid transporters; and an increasing number of bacterial archaeal, and eukaryotic proteins that have not yet been functionally characterized Sixty members of the glutamate transporter family,cere found ill the databases on the basis of sequence homology. The amino acid sequences of the carriers have diverged enormously. Homology between the members of the family is most apparent in a stretch of approximately 150 residues in the C-terminal part of the proteins. This region contains four reasonably well-conserved sequence motifs, all of which have been suggested to be part of the translocation pore or substrate binding site. Phylogenetic analysis of the C-terminal stretch revealed the presence of five subfamilies with characterized members: (i) the eukaryotic glutamate transporters, (ii) the bacterial glutamate transporters, (iii) the eukaryotic neutral-amino-acid transporters, (iv) the bacterial C-4-dicarboxylate transporters, and (v) the bacterial serine transporters. A number of other subfamilies that do not contain characterized members have been defined. In contrast to their amino acid sequences, the hydropathy profiles of the members of the family are extremely well conserved. Analysis of the hydropathy profiles has suggested that the glutamate transporters have a global structure that is unique among secondary transporters Experimentally, the unique structure of the transporters was recently confirmed by membrane topology studies. Although there is still controversy about part of the topology, the most likely model predicts the presence of eight membrane-spanning alpha-helices and a loop-pore structure which is unique among secondary transporters brit may resemble loop-pores found in ion channels. A second distinctive structural feature is the presence of a highly amphipathic membrane-spanning helix that provides a hydrophilic path through the membrane. Recent data from analysis of site-directed mutants and studies on the mechanism and pharmacology of the transporters are discussed in relation to the structural model.</p

Yeast Mitochondrial ADP/ATP Carriers Are Monomeric in Detergents as Demonstrated by Differential Affinity Purification

Most mitochondrial carriers carry out equimolar exchange of substrates and they are believed widely to exist as homo-dimers. Here we show by differential tagging that the yeast mitochondrial ADP/ATP carrier AAC2 is a monomer in mild detergents. Carriers with and without six-histidine or hemagglutinin tags were co-expressed in defined molar ratios in yeast mitochondrial membranes. Their specific transport activity was unaffected by tagging or by co-expression. The co-expressed carriers were extracted from the membranes with mild detergents and purified rapidly by affinity chromatography. All of the untagged carriers were in the flow-through of the affinity column, whereas all of the tagged carriers bound to the column and were eluted subsequently, showing that stable dimers, consisting of associated tagged and untagged carriers, were not present. The specific inhibitors carboxyatractyloside and bongkrekic acid and the substrates ADP, ATP and ADP plus ATP were added during the experiments to determine whether lack of association might have been caused by carriers being prevented from cycling through the various states in the transport cycle where dimers might form. All of the protein was accounted for, but stable dimers were not detected in any of these conditions, showing that yeast ADP/ATP carriers are monomeric in detergents in agreement with their hydrodynamic properties and with their structure. Since strong interactions between monomers were not observed in any part of the transport cycle, it is highly unlikely that the carriers function cooperatively. Therefore, transport mechanisms need to be considered in which the carrier is operational as a monomer

Structural divergence of paralogous S components from ECF-type ABC transporters

Energy coupling factor (ECF) proteins are ATP-binding cassette transporters involved in the import of micronutrients in prokaryotes. They consist of two nucleotide-binding subunits and the integral membrane subunit EcfT, which together form the ECF module and a second integral membrane subunit that captures the substrate (the S component). Different S components, unrelated in sequence and specific for different ligands, can interact with the same ECF module. Here, we present a high-resolution crystal structure at 2.1 Å of the biotin-specific S component BioY from Lactococcus lactis. BioY shares only 16% sequence identity with the thiamin-specific S component ThiT from the same organism, of which we recently solved a crystal structure. Consistent with the lack of sequence similarity, BioY and ThiT display large structural differences (rmsd = 5.1 Å), but the divergence is not equally distributed over the molecules: The S components contain a structurally conserved N-terminal domain that is involved in the interaction with the ECF module and a highly divergent C-terminal domain that binds the substrate. The domain structure explains howthe S components with large overall structural differences can interact with the same ECF module while at the same time specifically bind very different substrates with subnanomolar affinity. Solitary BioY (in the absence of the ECF module) is monomeric in detergent solution and binds D-biotin with a high affinity but does not transport the substrate across the membrane.