Transmembrane gate movements in the type II ATP-binding cassette (ABC) importer BtuCD-F during nucleotide cycle

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.     

Small Substrate Transport and Mechanism of a Molybdate ATP Binding Cassette Transporter in a Lipid Environment

Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria.     

Conformational Cycle of the Vitamin B12 ABC Importer in Liposomes Detected by Double Electron-Electron Resonance (DEER)

Double electron-electron resonance is used here to investigate intermediates of the transport cycle of the Escherichia coli vitamin B₁₂ ATP-binding cassette importer BtuCD-F. Previously, we showed the ATP-induced opening of the cytoplasmic gate I in TM5 helices, later confirmed by the AMP-PNP-bound BtuCD-F crystal structure. Here, other key residues are analyzed in TM10 helices (positions 307 and 322) and in the cytoplasmic gate II, i.e. the loop between TM2 and TM3 (positions 82 and 85). Without BtuF, binding of ATP induces detectable changes at positions 307 and 85 in BtuCD in liposomes. Together with BtuF, ATP triggers the closure of the cytoplasmic gate II in liposomes (reported by both positions 82 and 85). This forms a sealed cavity in the translocation channel in agreement with the AMP-PNP·BtuCD-F x-ray structure. When vitamin B₁₂ and AMP-PNP are simultaneously present, the extent of complex formation is reduced, but the short 82–82 interspin distance detected indicates that the substrate does not affect the closed conformation of this gate. The existence of the BtuCD-F complex under these conditions is verified with spectroscopically orthogonal nitroxide and Gd(III)-based labels. The cytoplasmic gate II remains closed also in the vanadate-trapped state, but it reopens in the ADP-bound state of the complex. Therefore, we suggest that the substrate likely trapped in ATP·BtuCD-F can be released after ATP hydrolysis but before the occluded ADP-bound conformation is reached.     

ATP binding cassette importers in eukaryotic organisms

ATP-binding cassette (ABC) transporters are ubiquitous across all realms of life. Dogma suggests that bacterial ABC transporters include both importers and exporters, whilst eukaryotic members of this family are solely exporters, implying that ABC import function was lost during evolution. This view is being challenged, for example energy-coupling factor (ECF)-type ABC importers appear to fulfil important roles in both algae and plants where they form the ABCI sub-family. Herein we discuss whether bacterial Type I and Type II ABC importers also made the transition into extant eukaryotes. Various studies suggest that Type I importers exist in algae and the liverwort family of primitive non-vascular plants, but not in higher plants. The existence of eukaryotic Type II importers is also supported: a transmembrane protein homologous to vitamin B12 import system transmembrane protein (BtuC), hemin transport system transmembrane protein (HmuU) and high-affinity zinc uptake system membrane protein (ZnuB) is present in the Cyanophora paradoxa genome. This protein has homologs within the genomes of red algae. Furthermore, its candidate nucleotide-binding domain (NBD) shows closest similarity to other bacterial Type II importer NBDs such as BtuD. Functional studies suggest that Type I importers have roles in maintaining sulphate levels in the chloroplast, whilst Type II importers probably act as importers of Mn²⁺ or Zn²⁺, as inferred by comparisons with bacterial homologs. Possible explanations for the presence of these transporters in simple plants, but not in other eukaryotic organisms, are considered. In order to utilise the existing nomenclature for eukaryotic ABC proteins, we propose that eukaryotic Type I and II importers be classified as ABCJ and ABCK transporters, respectively.     

Asymmetric Conformational Flexibility in the ATP-Binding Cassette Transporter HI1470/1

Putative metal-chelate-type ABC transporter HI1470/1 is homologous with vitamin B₁₂ importer BtuCD but exhibits a distinct inward-facing conformation in contrast to the outward-facing conformation of BtuCD. Normal-mode analysis of HI1470/1 reveals the intrinsic asymmetric conformational flexibility in this transporter and demonstrates that the transition from the inward-facing to the outward-facing conformation is realized through the asymmetric motion of individual subunits of the transporter. This analysis suggests that the asymmetric arrangement of the BtuC dimer in the crystal structure of the BtuCD-F complex represents an intermediate state relating HI1470/1 and BtuCD. Furthermore, a twisting motion between transmembrane domains and nucleotide-binding domains encoded in the lowest-frequency normal mode of this type of importer is found to contribute to the conformational transitions during the whole cycle of substrate transportation. A more complete translocation mechanism of the BtuCD type importer is proposed.     

In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes

Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC₂; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC₂, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg²⁺. We were able to purify a full complex, RbsABC₂, in the presence of stable, transition state mimics (ATP, Mg²⁺, and VO₄); a RbsAC complex in the presence of ADP and Mg²⁺; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC₂ shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.     

Distinct gate conformations of the ABC transporter BtuCD revealed by electron spin resonance spectroscopy and chemical cross-linking

BtuCD is a type II ABC importer that catalyzes the translocation of vitamin B12 from the periplasm into the cytoplasm of Escherichia coli. Crystal structures of BtuCD and the related HiF (or Hi1470/71) protein from Haemophilus influenzae have revealed distinct conformations of the transmembrane domains that form inner and outer gates. We used electron spin resonance spectroscopy to study the reaction cycle of BtuCD after labeling the protein at residues located at these gates. The results suggest that BtuCD as a prototype type II ABC importer may have a mechanism that is distinct from that of ABC exporters such as Sav1866 or type I ABC importers such as those specific for molybdate (ModBC) or maltose (MalFGK).

Structured summary

MINT-6803800: btuF (uniprotkb: P37028), btuC (uniprotkb:P06609) and btuD (uniprotkb:P06611) physically interact (MI:0218) by molecular sieving (MI:0071)     

The conformational transition pathways of ATP-binding cassette transporter BtuCD revealed by targeted molecular dynamics simulation

BtuCD is a member of the ATP-binding cassette transporters in Escherichia coli that imports vitamin B(12) into the cell by utilizing the energy of ATP hydrolysis. Crystal structures of BtuCD and its homologous protein HI1470/1 in various conformational states support the "alternating access" mechanism which proposes the conformational transitions of the substrate translocation pathway at transmembrane domain (TMD) between the outward-facing and inward-facing states. The conformational transition at TMD is assumed to couple with the movement of the cytoplasmic nucleotide-binding domains (NBDs) driven by ATP hydrolysis/binding. In this study, we performed targeted molecular dynamics (MD) simulations to explore the atomic details of the conformational transitions of BtuCD importer. The outward-facing to inward-facing (O→I) transition was found to be initiated by the conformational movement of NBDs. The subsequent reorientation of the substrate translocation pathway at TMD began with the closing of the periplasmic gate, followed by the opening of the cytoplamic gate in the last stage of the conformational transition due to the extensive hydrophobic interactions at this region, consistent with the functional requirement of unidirectional transport of the substrates. The reverse inward-facing to outward-facing (I→O) transition was found to exhibit intrinsic diversity of the conformational transition pathways and significant structural asymmetry, suggesting that the asymmetric crystal structure of BtuCD-F is an intermediate state in this process.     

Conformational coupling of the nucleotide-binding and the transmembrane domains in ABC transporters

Basic architecture of ABC transporters includes two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Although the transport process takes place in the TMDs, which provide the substrate translocation pathway across the cell membrane and control its accessibility between the two sides of the membrane, the energy required for the process is provided by conformational changes induced in the NBDs by binding and hydrolysis of ATP. Nucleotide-dependent conformational changes in the NBDs, therefore, need to be coupled to structural changes in the TMDs. Using molecular dynamics simulations, we have investigated the structural elements involved in the conformational coupling between the NBDs and the TMDs in the Escherichia coli maltose transporter, an ABC importer for which an intact structure is available both in inward-facing and outward-facing conformations. The prevailing model of coupling is primarily based on a single structural motif, known as the coupling helices, as the main structural element for the NBD-TMD coupling. Surprisingly, we find that in the absence of the NBDs the coupling helices can be conformationally decoupled from the rest of the TMDs, despite their covalent connection. That is, the structural integrity of the coupling helices and their tight coupling to the core of the TMDs rely on the contacts provided by the NBDs. Based on the conformational and dynamical analysis of the simulation trajectories, we propose that the core coupling elements in the maltose transporter involve contributions from several structural motifs located at the NBD-TMD interface, namely, the EAA loops from the TMDs, and the Q-loop and the ENI motifs from the NBDs. These three structural motifs in small ABC importers show a high degree of correlation in motion and mediate the necessary conformational coupling between the core of TMDs and the helical subdomains of NBDs. A comprehensive analysis of the structurally known ABC transporters shows a high degree of conservation of the identified 3-motif coupling elements only in the subfamily of small ABC importers, suggesting a distinct mode of NBD-TMD coupling from the other two major ABC transporter folds, namely large ABC importers and ABC exporters.     

Diversity in ABC transporters: Type I,II and III importers

Abstract

ATP-binding cassette transporters are multi-subunit membrane pumps that transport substrates across membranes. While significant in the transport process, transporter architecture exhibits a range of diversity that we are only beginning to recognize. This divergence may provide insight into the mechanisms of substrate transport and homeostasis. Until recently, ABC importers have been classified into two types, but with the emergence of energy-coupling factor (ECF) transporters there are potentially three types of ABC importers. In this review, we summarize an expansive body of research on the three types of importers with an emphasis on the basics that underlie ABC importers, such as structure, subunit composition and mechanism.     

Energy Coupling Efficiency in the Type I ABC Transporter GlnPQ

Solute transport via ATP binding cassette (ABC) importers involves receptor-mediated substrate binding, which is followed by ATP-driven translocation of the substrate across the membrane. How these steps are exactly initiated and coupled, and how much ATP it takes to complete a full transport cycle, are subject of debate. Here, we reconstitute the ABC importer GlnPQ in nanodiscs and in proteoliposomes and determine substrate-(in)dependent ATP hydrolysis and transmembrane transport. We determined the conformational states of the substrate-binding domains (SBDs) by single-molecule Förster resonance energy transfer measurements. We find that the basal ATPase activity (ATP hydrolysis in the absence of substrate) is mainly caused by the docking of the closed-unliganded state of the SBDs onto the transporter domain of GlnPQ and that, unlike glutamine, arginine binds both SBDs but does not trigger their closing. Furthermore, comparison of the ATPase activity in nanodiscs with glutamine transport in proteoliposomes shows that the stoichiometry of ATP per substrate is close to two. These findings help understand the mechanism of transport and the energy coupling efficiency in ABC transporters with covalently linked SBDs, which may aid our understanding of Type I ABC importers in general.     

A Multitask ATPase Serving Different ABC-Type Sugar Importers in Bacillus subtilis

Bacillus subtilis is able to utilize arabinopolysaccharides derived from plant biomass. Here, by combining genetic and physiological analyses we characterize the AraNPQ importer and identify primary and secondary transporters of B. subtilis involved in the uptake of arabinosaccharides. We show that the ABC-type importer AraNPQ is involved in the uptake of α-1,5-arabinooligosaccharides, at least up to four l-arabinosyl units. Although this system is the key transporter for α-1,5-arabinotriose and α-1,5-arabinotetraose, the results indicate that α-1,5-arabinobiose also is translocated by the secondary transporter AraE. This broad-specificity proton symporter is the major transporter for arabinose and also is accountable for the uptake of xylose and galactose. In addition, MsmX is shown to be the ATPase that energizes the incomplete AraNPQ importer. Furthermore, the results suggest the existence of at least one more unidentified MsmX-dependent ABC importer responsible for the uptake of nonlinear α-1,2- and α-1,3-arabinooligosaccharides. This study assigns MsmX as a multipurpose B. subtilis ATPase required to energize different saccharide transporters, the arabinooligosaccharide-specific AraNPQ-MsmX system, a putative MsmX-dependent ABC transporter specific for nonlinear arabinooligosaccharides, and the previously characterized maltodextrin-specific MdxEFG-MsmX system.Transport across biological membranes is a fundamental process for life and is accomplished by channels, primary and secondary transporters, and group translocators (19). ATP-binding cassette (ABC) transporters constitute one of the largest families of translocation facilitators and are distributed across all domains of life. Although they are involved in a variety of distinct processes, such as nutrient uptake, resistance to antibiotics and other drugs, lipid trafficking, cell division, sporulation, immune response, and pathogenesis, all ABC transporters, regardless of the polarity of transport (exporters and importers), share a structure and a general mechanism (reviewed in references 3 and 8). Their organization comprises two transmembrane domains (TMDs) coupled to two cytosolic nucleotide-binding domains (NBDs), or ATP-binding cassettes, responsible for ATP binding and hydrolysis-driven conformational changes.The majority of the eukaryotic ABC transporters are exporters facilitating translocation from the cytoplasm. In contrast, prokaryotic ABC permeases are involved mainly in the import of nutrients, vitamins, and trace elements (3, 6, 8). Canonical bacterial ABC importers are dependent predominantly on high-affinity substrate-binding proteins (BPDs) that capture the substrate and deliver it to the transporter. The canonical maltose/maltodextrin importer MalEFGK₂ of Escherichia coli/Salmonella enterica serovar Typhimurium (5) is one of the most-characterized members of this superfamily of translocation facilitators, serving as model for ABC importers in general (14, 15). In Gram-negative bacteria, BPDs are proteins located in the periplasmic space between the inner and outer membranes. In Gram-positive organisms, which are devoid of this type of cellular compartment, BPDs often are lipoproteins that are anchored to the extracellular side of the cytoplasmic membrane via its N-terminal domain (3 and 8 and references therein).In nature, the major source of carbohydrates for microorganisms to utilize is plant biomass. Thus, in their natural habitat, such as soil, aquatic environments, or animal digestive tracts, bacteria secrete a vast number of polysaccharolytic enzymes for the degradation of plant-derived polysaccharides. The resulting mono-, di-, and oligosaccharides enter the cell mainly through specific ABC transporters. The number of well-characterized ABC transporters devoted to the uptake of products resulting from the degradation of hemicellulose is very scarce (6), and in Gram-positive organisms only two systems, BxlEFG of Streptomyces thermoviolaceus (31) and XynEFG (29) of Geobacillus staerothermophilus, both dedicated to the transport of xylodextrins, have been characterized in detail.An in silico analysis of the Bacillus subtilis genome estimated the existence of at least 78 ABC transporters based on the identification of 86 NBDs in 78 proteins, 103 MSD proteins, and 37 BPD proteins, which account for about 5% of the protein-coding genes of this model organism (17). At least 10 ABC systems are predicted to be involved in the uptake of sugars (20). One of these ABC importers, AraNPQ, is clustered together with genes encoding enzymes involved in arabinose catabolism and the degradation of arabinooligosaccharides in a large operon, araABDLMNPQ-abfA (23). AraN is the BPD, and AraP and AraQ are the TMDs. This transporter, which lacks the NBD protein partner, was proposed to be involved in the uptake of arabinose oligomers mainly by genomic context and in silico analysis (10, 11, 23).Here, by combining genetic and physiological analyses, we characterize the AraNPQ importer and identify primary and secondary transporters of B. subtilis involved in the uptake of arabinosaccharides. Furthermore, this study assigns the role of MsmX as a multipurpose B. subtilis ATPase required to energize different saccharide transporters.     

Selective vitamin B12 malabsorption in two siblings

Two siblings with megaloblastic anemia responsive to parenteral vitamin B₁₂ were studied to elucidate the cause of the B₁₂ deficiency. Gastric juice from both contained acid and functional intrinsic factor. Serum contained transcobalamin II and lacked antibodies to intrinsic factor. Schilling tests showed vitamin B₁₂ malabsorption uncorrected by hog intrinsic factor or pancreatic extract. Other parameters of small intestinal function were normal. Proteinuria was initially present in both but cleared in one following treatment with B₁₂. These patients with “familial selective vitamin B₁₂ malabsorption” are the first reported from Canada. Only 37 cases have been reported in the world literature to date.     

The conformational coupling and translocation mechanism of vitamin B12 ATP-binding cassette transporter BtuCD

ATP-binding cassette transporter BtuCD mediating vitamin B₁₂ uptake in Escherichia coli couples the energy of ATP hydrolysis to the translocation of vitamin B₁₂ across the membrane into the cell. Elastic normal mode analysis of BtuCD demonstrates that the simultaneous substrate trapping at periplasmic cavity and ATP binding at the ATP-binding cassette (BtuD) dimer proceeds readily along the lowest energy pathway. The transport power stroke is attributed to ATP-hydrolysis-induced opening of the nucleotide-binding domain dimer, which is coupled to conformational rearrangement of transmembrane domain (BtuC) helices leading to the closing at the periplasmic side and opening at the cytoplasmic gate. Simultaneous hydrolysis of two ATP is supported by the fact that antisymmetric movement of BtuD dimer implying alternating hydrolysis cannot induce effective conformational change of the translocation pathway. A plausible mechanism of translocation cycle is proposed in which the possible effect of the association of periplasmic binding protein BtuF to the transporter is also considered.     

Transmembrane Signaling in the Maltose ABC Transporter MalFGK2-E: PERIPLASMIC MalF-P2 LOOP COMMUNICATES SUBSTRATE AVAILABILITY TO THE ATP-BOUND MalK DIMER*

ABC transporters are ubiquitous membrane proteins that translocate solutes across biological membranes at the expense of ATP. In prokaryotic ABC importers, the extracytoplasmic anchoring of the substrate-binding protein (receptor) is emerging as a key determinant for the structural rearrangements in the cytoplasmically exposed ATP-binding cassette domains and in the transmembrane gates during the nucleotide cycle. Here the molecular mechanism of such signaling events was addressed by electron paramagnetic resonance spectroscopy of spin-labeled ATP-binding cassette maltose transporter variants (MalFGK₂-E). A series of doubly spin-labeled mutants in the MalF-P2 domain involving positions 92, 205, 239, 252, and 273 and one triple mutant labeled at positions 205/252 in P2 and 83 in the Q-loop of MalK were assayed. The EPR data revealed that the substrate-binding protein MalE is bound to the transporter throughout the transport cycle. Concomitantly with the three conformations of the ATP-binding cassette MalK₂, three functionally relevant conformations are found also in the periplasmic MalF-P2 loop, strictly dependent on cytoplasmic nucleotide binding and periplasmic docking of liganded MalE to MalFG. The reciprocal communication across the membrane unveiled here gives first insights into the stimulatory effect of MalE on the ATPase activity, and it is suggested to be an important mechanistic feature of receptor-coupled ABC transporters.     

Role of Third Serum Vitamin B12 Binding Protein in Vitamin B12 Transport

A third vitamin B₁₂ binding protein present in normal serum has been shown to participate in transport of labelled vitamin B₁₂ absorbed from the gut. All three vitamin B₁₂ binding proteins in serum were labelled at the same time after oral administration of vitamin B₁₂, implying that “free” vitamin B₁₂ reached the portal blood from the gut mucosa.     

Whole-Genome Survey of the Putative ATP-Binding Cassette Transporter Family Genes in Vitis vinifera

The ATP-binding cassette (ABC) protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 “full-size,” 41 “half-size,” and 15 “soluble” putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog) and ABCC (multidrug resistance-associated protein). We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera.     

Simulation of the coupling between nucleotide binding and transmembrane domains in the ATP binding cassette transporter BtuCD

The nucleotide-induced structural rearrangements in ATP binding cassette (ABC) transporters, leading to substrate translocation, are largely unknown. We have modeled nucleotide binding and release in the vitamin B(12) importer BtuCD using perturbed elastic network calculations and biased molecular dynamics simulations. Both models predict that nucleotide release decreases the tilt between the two transmembrane domains and opens the cytoplasmic gate. Nucleotide binding has the opposite effect. The observed coupling may be relevant for all ABC transporters because of the conservation of nucleotide binding domains and the shared role of ATP in ABC transporters. The rearrangements in the cytoplasmic gate region do not provide enough space for B(12) to diffuse from the transporter pore into the cytoplasm, which could suggest that peristaltic forces are needed to exclude B(12) from the transporter pore.     

Thermodynamics of ABC transporters

ABC transporters form the largest of all transporter families, and their structural study has made tremendous progress over recent years. However, despite such advances, the precise mechanisms that determine the energy-coupling between ATP hydrolysis and the conformational changes following substrate binding remain to be elucidated. Here, we present our thermodynamic analysis for both ABC importers and exporters, and introduce the two new concepts of differential-binding energy and elastic conformational energy into the discussion.We hope that the structural analysis of ABC transporters will henceforth take thermodynamic aspects of transport mechanisms into account as well.     

Titratable transmembrane residues and a hydrophobic plug are essential for manganese import via the Bacillus anthracis ABC transporter MntBC-A

All extant life forms require trace transition metals (e.g., Fe^2/3+, Cu^1/2+, and Mn²⁺) to survive. However, as these are environmentally scarce, organisms have evolved sophisticated metal uptake machineries. In bacteria, high-affinity import of transition metals is predominantly mediated by ABC transporters. During bacterial infection, sequestration of metal by the host further limits the availability of these ions, and accordingly, bacterial ABC transporters (importers) of metals are key virulence determinants. However, the structure–function relationships of these metal transporters have not been fully elucidated. Here, we used metal-sensitivity assays, advanced structural modeling, and enzymatic assays to study the ABC transporter MntBC-A, a virulence determinant of the bacterial human pathogen Bacillus anthracis. We find that despite its broad metal-recognition profile, MntBC-A imports only manganese, whereas zinc can function as a high-affinity inhibitor of MntBC-A. Computational analysis shows that the transmembrane metal permeation pathway is lined with six titratable residues that can coordinate the positively charged metal, and mutagenesis studies show that they are essential for manganese transport. Modeling suggests that access to these titratable residues is blocked by a ladder of hydrophobic residues, and ATP-driven conformational changes open and close this hydrophobic seal to permit metal binding and release. The conservation of this arrangement of titratable and hydrophobic residues among ABC transporters of transition metals suggests a common mechanism. These findings advance our understanding of transmembrane metal recognition and permeation and may aid the design and development of novel antibacterial agents.