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

Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment

Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each protomer functioning independently by an elevator-type mechanism, in which a mobile transport domain alternates between inward- and outward-oriented states. Using single-particle cryo-EM we have determined five structures of the glutamate transporter homologue GltTk, a Na+- L-aspartate symporter, embedded in lipid nanodiscs. Dependent on the substrate concentrations used, the protomers of the trimer adopt a variety of asymmetrical conformations, consistent with the independent movement. Six of the 15 resolved protomers are in a hitherto elusive state of the transport cycle in which the inward-facing transporters are loaded with Na+ ions. These structures explain how substrate-leakage is prevented – a strict requirement for coupled transport. The belt protein of the lipid nanodiscs bends around the inward oriented protomers, suggesting that membrane deformations occur during transport

Elevator-type mechanisms of membrane transport

Membrane transporters are integral membrane proteins that mediate the passage of solutes across lipid bilayers. These proteins undergo conformational transitions between outward- and inward-facing states, which lead to alternating access of the substrate-binding site to the aqueous environment on either side of the membrane. Dozens of different transporter families have evolved, providing a wide variety of structural solutions to achieve alternating access. A sub-set of structurally diverse transporters operate by mechanisms that are collectively named 'elevator-type'. These transporters have one common characteristic: they contain a distinct protein domain that slides across the membrane as a rigid body, and in doing so it 'drags" the transported substrate along. Analysis of the global conformational changes that take place in membrane transporters using elevator-type mechanisms reveals that elevator-type movements can be achieved in more than one way. Molecular dynamics simulations and experimental data help to understand how lipid bilayer properties may affect elevator movements and vice versa

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