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.     

Bacterial ATP-driven transporters of transition metals: physiological roles, mechanisms of action, and roles in bacterial virulence

Maintaining adequate intracellular levels of transition metals is fundamental to the survival of all organisms. While all transition metals are toxic at elevated intracellular concentrations, metals such as iron, zinc, copper, and manganese are essential to many cellular functions. In prokaryotes, the concerted action of a battery of membrane-embedded transport proteins controls a delicate balance between sufficient acquisition and overload. Representatives from all major families of transporters participate in this task, including ion-gradient driven systems and ATP-utilizing pumps. P-type ATPases and ABC transporters both utilize the free energy of ATP hydrolysis to drive transport. Each of these very different families of transport proteins has a distinct role in maintaining transition metal homeostasis: P-type ATPases prevent intracellular overloading of both essential and toxic metals through efflux while ABC transporters import solely the essential ones. In the present review we discuss how each system is adapted to perform its specific task from mechanistic and structural perspectives. Despite the mechanistic and structural differences between P-type ATPases and ABC transporters, there is one important commonality: in many clinically relevant bacterial pathogens, transporters of transition metals are essential for virulence. Here we present several such examples and discuss how these may be exploited for future antibacterial drug development.     

A tetrahedral coordination of Zinc during transmembrane transport by P-type Zn2+-ATPases

Zn²⁺ is an essential transition metal required in trace amounts by all living organisms. However, metal excess is cytotoxic and leads to cell damage. Cells rely on transmembrane transporters, with the assistance of other proteins, to establish and maintain Zn²⁺ homeostasis. Metal coordination during transport is key to specific transport and unidirectional translocation without the backward release of free metal. The coordination details of Zn²⁺ at the transmembrane metal binding site responsible for transport have now been established. Escherichia coli ZntA is a well-characterized Zn²⁺-ATPase responsible for intracellular Zn²⁺ efflux. A truncated form of the protein lacking regulatory metal sites and retaining the transport site was constructed. Metrical parameters of the metal–ligand coordination geometry for the zinc bound isolated form were characterized using x-ray absorption spectroscopy (XAS). Our data support a nearest neighbor ligand environment of (O/N)₂S₂ that is compatible with the proposed invariant metal coordinating residues present in the transmembrane region. This ligand identification and the calculated bond lengths support a tetrahedral coordination geometry for Zn²⁺ bound to the TM-MBS of P-type ATPase transporters.     

The phage shock protein PspA facilitates divalent metal transport and is required for virulence of Salmonella enterica sv. Typhimurium

The phage shock protein (Psp) system is induced by extracytoplasmic stress and thought to be important for the maintenance of proton motive force. We investigated the contribution of PspA to Salmonella virulence. A pspA deletion mutation significantly attenuates the virulence of Salmonella enterica serovar Typhimurium following intraperitoneal inoculation of C3H/HeN (Ity^r) mice. PspA was found to be specifically required for virulence in mice expressing the natural resistance‐associated macrophage protein 1 (Nramp1) (Slc11a1) divalent metal transporter, which restricts microbial growth by limiting the availability of essential divalent metals within the phagosome. Salmonella competes with Nramp1 by expressing multiple metal uptake systems including the Nramp‐homologue MntH, the ABC transporter SitABCD and the ZIP family transporter ZupT. PspA was found to facilitate Mn²⁺ transport by MntH and SitABCD, as well as Zn²⁺ and Mn²⁺ transport by ZupT. In vitro uptake of ⁵⁴Mn²⁺ by MntH and ZupT was reduced in the absence of PspA. Transport‐deficient mutants exhibit reduced viability in the absence of PspA when grown under metal‐limited conditions. Moreover, the ZupT transporter is required for Salmonella enterica serovar Typhimurium virulence in Nramp1‐expressing mice. We propose that PspA promotes Salmonella virulence by maintaining proton motive force, which is required for the function of multiple transporters mediating bacterial divalent metal acquisition during infection.     

Nickel transport systems in microorganisms

The transition metal Ni is an essential cofactor for a number of enzymatic reactions in both prokaryotes and eukaryotes. Molecular analyses have revealed the existence of two major types of high-affinity Ni²⁺ transporters in bacteria. The Nik system of Escherichia coli is a member of the ABC transporter family and provides Ni²⁺ ion for the anaerobic biosynthesis of hydrogenases. The periplasmic binding protein of the transporter, NikA, is likely to play a dual role. It acts as the primary binder in the uptake process and is also involved in negative chemotaxis to escape Ni overload. Expression of the nik operon is controlled by the Ni-responsive repressor NikR, which shows functional similarity to the ferric ion uptake regulator Fur. The second type of Ni²⁺ transporter is represented by HoxN of Ralstonia eutropha, the prototype of a novel family of transition metal permeases. Members of this family have been identified in gram-negative and gram-positive bacteria and recently also in a fission yeast. They transport Ni²⁺ with very high affinity, but differ with regard to specificity. Site-directed mutagenesis experiments have identified residues that are essential for transport. Besides these uptake systems, different types of metal export systems, which prevent microorganisms from the toxic effects of Ni²⁺ at elevated intracellular concentrations, have also been described. Received: 14 July / Accepted: 8 October 1999     

Role of CFTR’s intrinsic adenylate kinase activity in gating of the Cl− channel

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl^?channel in the ATP-binding cassette (ABC) transporter protein family. CFTR features the modular design characteristic of ABC transporters, which includes two membrane-spanning domains forming the channel pore, and two ABC nucleotide-binding domains that interact with ATP and contain the enzymatic activity coupled to normal gating. Like other ABC transporters CFTR is an ATPase (ATP + H₂O → ADP + P_i). Recent work has shown that CFTR also possesses intrinsic adenylate kinase activity (ATP + AMP ? ADP + ADP). This finding raises important questions: How does AMP influence CFTR gating? Why does ADP inhibit CFTR current? Which enzymatic activity gates CFTR in vivo? Are there implications for other ABC transporters? This minireview attempts to shed light on these questions by summarizing recent advances in our understanding of the role of the CFTR adenylate kinase activity for channel gating.     

Solitary BioY Proteins Mediate Biotin Transport into Recombinant Escherichia coli

Energy-coupling factor (ECF) transporters form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homo- or hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotin-specific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, however, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Escherichia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was constructed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteobacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the recombinants, which was inferred from uptake assays with [³H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane.     

Planar substrate-binding site dictates the specificity of ECF-type nickel/cobalt transporters

The energy-coupling factor (ECF) transporters are multi-subunit protein complexes that mediate uptake of transition-metal ions and vitamins in about 50% of the prokaryotes, including bacteria and archaea. Biological and structural studies have been focused on ECF transporters for vitamins, but the molecular mechanism by which ECF systems transport metal ions from the environment remains unknown. Here we report the first crystal structure of a NikM, TtNikM2, the substrate-binding component (S component) of an ECF-type nickel transporter from Thermoanaerobacter tengcongensis. In contrast to the structures of the vitamin-specific S proteins with six transmembrane segments (TSs), TtNikM2 possesses an additional TS at its N-terminal region, resulting in an extracellular N-terminus. The highly conserved N-terminal loop inserts into the center of TtNikM2 and occludes a region corresponding to the substrate-binding sites of the vitamin-specific S components. Nickel binds to NikM via its coordination to four nitrogen atoms, which are derived from Met1, His2 and His67 residues. These nitrogen atoms form an approximately square-planar geometry, similar to that of the metal ion-binding sites in the amino-terminal Cu²⁺- and Ni²⁺-binding (ATCUN) motif. Replacements of residues in NikM contributing to nickel coordination compromised the Ni-transport activity. Furthermore, systematic quantum chemical investigation indicated that this geometry enables NikM to also selectively recognize Co²⁺. Indeed, the structure of TtNikM2 containing a bound Co²⁺ ion has almost no conformational change compared to the structure that contains a nickel ion. Together, our data reveal an evolutionarily conserved mechanism underlying the metal selectivity of EcfS proteins, and provide insights into the ion-translocation process mediated by ECF transporters.     

The ABC transporters in Candidatus Liberibacter asiaticus

Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a Gram‐negative bacterium and the pathogen of Citrus Greening disease (Huanglongbing, HLB). As a parasitic bacterium, Ca. L. asiaticus harbors ABC transporters that play important roles in exchanging chemical compounds between Ca. L. asiaticus and its host. Here, we analyzed all the ABC transporter‐related proteins in Ca. L. asiaticus. We identified 14 ABC transporter systems and predicted their structures and substrate specificities. In‐depth sequence and structure analysis including multiple sequence alignment, phylogenetic tree reconstruction, and structure comparison further support their function predictions. Our study shows that this bacterium could use these ABC transporters to import metabolites (amino acids and phosphates) and enzyme cofactors (choline, thiamine, iron, manganese, and zinc), resist to organic solvent, heavy metal, and lipid‐like drugs, maintain the composition of the outer membrane (OM), and secrete virulence factors. Although the features of most ABC systems could be deduced from the abundant experimental data on their orthologs, we reported several novel observations within ABC system proteins. Moreover, we identified seven nontransport ABC systems that are likely involved in virulence gene expression regulation, transposon excision regulation, and DNA repair. Our analysis reveals several candidates for further studies to understand and control the disease, including the type I virulence factor secretion system and its substrate that are likely related to Ca. L. asiaticus pathogenicity and the ABC transporter systems responsible for bacterial OM biosynthesis that are good drug targets. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Pseudomonas aeruginosa Cif Protein Enhances the Ubiquitination and Proteasomal Degradation of the Transporter Associated with Antigen Processing (TAP) and Reduces Major Histocompatibility Complex (MHC) Class I Antigen Presentation

Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8⁺ T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.     

Synthesis and antimalarial activity of metal complexes of cross-bridged tetraazamacrocyclic ligands

Using transition metals such as manganese(II), iron(II), cobalt(II), nickel(II), copper(II), and zinc(II), several new metal complexes of cross-bridged tetraazamacrocyclic chelators namely, cyclen- and cyclam-analogs with benzyl groups, were synthesized and screened for in vitro antimalarial activity against chloroquine-resistant (W2) and chloroquine-sensitive (D6) strains of Plasmodium falciparum. The metal-free chelators tested showed little or no antimalarial activity. All the metal complexes of the dibenzyl cross-bridged cyclam ligand exhibited potent antimalarial activity. The Mn²⁺ complex of this ligand was the most potent with IC₅₀s of 0.127 and 0.157 μM against the chloroquine-sensitive (D6) and chloroquine-resistant (W2) P. falciparum strains, respectively. In general, the dibenzyl hydrophobic ligands showed better anti-malarial activity compared to the activity of monobenzyl ligands, potentially because of their higher lipophilicity and thus better cell penetration ability. The higher antimalarial activity displayed by the manganese complex for the cyclam ligand in comparison to that of the cyclen, correlates with the larger pocket of cyclam compared to that of cyclen which produces a more stable complex with the Mn²⁺. Few of the Cu²⁺ and Fe²⁺ complexes also showed improvement in activity but Ni²⁺, Co²⁺ and Zn²⁺ complexes did not show any improvement in activity upon the metal-free ligands for anti-malarial development.     

Insight into the interaction of metal ions with TroA from Streptococcus suis

Background

The scavenging ability of sufficient divalent metal ions is pivotal for pathogenic bacteria to survive in the host. ATP-binding cassette (ABC)-type metal transporters provide a considerable amount of different transition metals for bacterial growth. TroA is a substrate binding protein for uptake of multiple metal ions. However, the function and structure of the TroA homologue from the epidemic Streptococcus suis isolates (SsTroA) have not been characterized.

Methodology/Principal Findings

Here we determined the crystal structure of SsTroA from a highly pathogenic streptococcal toxic shock syndrome (STSS)-causing Streptococcus suis in complex with zinc. Inductively coupled plasma mass spectrometry (ICP-MS) analysis revealed that apo-SsTroA binds Zn²⁺ and Mn²⁺. Both metals bind to SsTroA with nanomolar affinity and stabilize the protein against thermal unfolding. Zn²⁺ and Mn²⁺ induce distinct conformational changes in SsTroA compared with the apo form as confirmed by both circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra. NMR data also revealed that Zn²⁺/Mn²⁺ bind to SsTroA in either the same site or an adjacent region. Finally, we found that the folding of the metal-bound protein is more compact than the corresponding apoprotein.

Conclusions/Significance

Our findings reveal a mechanism for uptake of metal ions in S. suis and this mechanism provides a reasonable explanation as to how SsTroA operates in metal transport.     

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.     

The cation diffusion facilitator protein MamM's cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2+

Cation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain, in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal or multiple metals from the cytoplasm, and it is not known whether the CTD takes an active regulatory role in metal recognition and discrimination during cation transport. Here, the model CDF protein MamM, an iron transporter from magnetotactic bacteria, was used to probe the role of the CTD in metal recognition and selectivity. Using a combination of biophysical and structural approaches, the binding of different metals to MamM CTD was characterized. Results reveal that different metals bind distinctively to MamM CTD in terms of their binding sites, thermodynamics, and binding-dependent conformations, both in crystal form and in solution, which suggests a varying level of functional discrimination between CDF domains. Furthermore, these results provide the first direct evidence that CDF CTDs play a role in metal selectivity. We demonstrate that MamM''s CTD can discriminate against Mn²⁺, supporting its postulated role in preventing magnetite formation poisoning in magnetotactic bacteria via Mn²⁺ incorporation.     

Molecular modeling of the human multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is a 190 kDa member of the ATP-binding cassette (ABC) superfamily of transmembrane transporters that is clinically relevant for its ability to confer multidrug resistance by actively effluxing anticancer drugs. Knowledge of the atomic structure of MRP1 is needed to elucidate its transport mechanism, but only low resolution structural data are currently available. Consequently, comparative modeling has been used to generate models of human MRP1 based on the crystal structure of the ABC transporter Sav1866 from Staphylococcus aureus. In these Sav1866-based models, the arrangement of transmembrane helices differs strikingly from earlier models of MRP1 based on the structure of the bacterial lipid transporter MsbA, both with respect to packing of the twelve helices and their interactions with the nucleotide binding domains. The functional importance of Tyr³²⁴ in transmembrane helix 6 predicted to project into the substrate translocation pathway was investigated.     

Transcriptome-wide identification and expression analyses of ABC transporters in dwarf polish wheat under metal stresses

ABC transporters, which comprise one of the largest protein families, are involved in maintaining osmotic homeostasis, nutrient uptake, pathogen resistance, and metal tolerance. In this study, 30 ABC genes in dwarf polish wheat were characterized and classified into seven subfamilies (ABCA - ABCG). Among them, 24 ABC transporters were newly found in wheat. The expressions of 13 ABC genes in roots and leaves under six metal stresses were also analyzed. All these genes were differentially regulated by Cd (except ABCE2, ABCF4, and ABCF6 in roots), suggesting that these genes participate in Cd transport, sequestration, or uptake. These genes were also differentially regulated by other metals including Cu, Mg, Zn, Fe, and Ni. Results suggest that the expressions of ABC transporters in dwarf polish wheat played important roles in metal transport and detoxification.     

Identification of functional amino acids in the Nramp family by a combination of evolutionary analysis and biophysical studies of metal and proton cotransport in vivo

The natural resistance-associated macrophage protein (Nramp) family is functionally conserved in bacteria and eukarya; Nramp homologues function as proton-dependent membrane transporters of divalent metals. Sequence analyses indicate that five phylogenetic groups comprise the Nramp family, three bacterial and two eukaryotic, which are distinct from a more distantly related group of microbial sequences (Nramp outgroup). The Nramp family and outgroup share many conserved residues, suggesting they derived from a common ancestor and raising the possibility that the residues invariant in the Nramp family that correspond to residues which are different but also conserved in the outgroup represent candidate sites of functional divergence of the Nramp family. Four Nramp family-specific residues were identified within transmembrane domains 1, 6, and 11, and replaced by the corresponding invariant outgroup residues in the Escherichia coli Nramp ortholog (the proton-dependent manganese transporter, MntH of group A, EcoliA). The resulting mutants (Asp(34)Gly, Asn(37)Thr, His(211)Tyr, and Asn(401)Gly) were tested for both divalent metal uptake and proton transport; quasi-simultaneous analyses of uptake of metals and protons revealed for the first time protons and metals cotransport by a bacterial Nramp homologue. Additional mutations were studied for comparison (Asp(34)Asn, Asn(37)Asp and Asn(37)Val, Asn(401)Thr, His(211)Ala, His(216)Ala, and His(216)Arg). EcoliA activity was impaired after each of the Nramp/outgroup substitutions, as well as after more conservative replacements, showing that the tested sites are all important for metal uptake and metal-dependent H(+) transport. It is proposed that co-occurrence of these four Nramp-specific transmembrane residues may have contributed to the emergence of this family of metal and proton cotransporters.     

Transition metal cation inhibition of Mycobacterium tuberculosis esterase RV0045C

Mycobacterium tuberculosis virulence is highly metal‐dependent with metal availability modulating the shift from the dormant to active states of M. tuberculosis infection. Rv0045c from M. tuberculosis is a proposed metabolic serine hydrolase whose folded stability is dependent on divalent metal concentration. Herein, we measured the divalent metal inhibition profile of the enzymatic activity of Rv0045c and found specific divalent transition metal cations (Cu²⁺ ≥ Zn²⁺ > Ni²⁺ > Co²⁺) strongly inhibited its enzymatic activity. The metal cations bind allosterically, largely affecting values for k _cat rather than K _M. Removal of the artificial N‐terminal 6xHis‐tag did not change the metal‐dependent inhibition, indicating that the allosteric inhibition site is native to Rv0045c. To isolate the site of this allosteric regulation in Rv0045c, the structures of Rv0045c were determined at 1.8 Å and 2.0 Å resolution in the presence and absence of Zn²⁺ with each structure containing a previously unresolved dynamic loop spanning the binding pocket. Through the combination of structural analysis with and without zinc and targeted mutagenesis, this metal‐dependent inhibition was traced to multiple chelating residues (H202A/E204A) on a flexible loop, suggesting dynamic allosteric regulation of Rv0045c by divalent metals. Although serine hydrolases like Rv0045c are a large and diverse enzyme superfamily, this is the first structural confirmation of allosteric regulation of their enzymatic activity by divalent metals.     

Structural and Functional Characterization of an Orphan ATP-Binding Cassette ATPase Involved in Manganese Utilization and Tolerance in Leptospira spp.

Pathogenic Leptospira species are the etiological agents of the widespread zoonotic disease leptospirosis. Most organisms, including Leptospira, require divalent cations for proper growth, but because of their high reactivity, these metals are toxic at high concentrations. Therefore, bacteria have acquired strategies to maintain metal homeostasis, such as metal import and efflux. By screening Leptospira biflexa transposon mutants for their ability to use Mn²⁺, we have identified a gene encoding a putative orphan ATP-binding cassette (ABC) ATPase of unknown function. Inactivation of this gene in both L. biflexa and L. interrogans strains led to mutants unable to grow in medium in which iron was replaced by Mn²⁺, suggesting an involvement of this ABC ATPase in divalent cation uptake. A mutation in this ATPase-coding gene increased susceptibility to Mn²⁺ toxicity. Recombinant ABC ATPase of the pathogen L. interrogans exhibited Mg²⁺-dependent ATPase activity involving a P-loop motif. The structure of this ATPase was solved from a crystal containing two monomers in the asymmetric unit. Each monomer adopted a canonical two-subdomain organization of the ABC ATPase fold with an α/β subdomain containing the Walker motifs and an α subdomain containing the ABC signature motif (LSSGE). The two monomers were arranged in a head-to-tail orientation, forming a V-shaped particle with all the conserved ABC motifs at the dimer interface, similar to functional ABC ATPases. These results provide the first structural and functional characterization of a leptospiral ABC ATPase.     

A sulfur-based transport pathway in Cu+-ATPases

Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. P_IB-type Cu⁺-ATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu⁺ across cellular membranes. Crystal structures of a copper-free Cu⁺-ATPase are available, but the mechanism of Cu⁺ recognition, binding, and translocation remains elusive. Through X-ray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solid-supported membranes using wild-type and mutant forms of the Legionella pneumophila Cu⁺-ATPase (LpCopA), we identify a sulfur-lined metal transport pathway. Structural analysis indicates that Cu⁺ is bound at a high-affinity transmembrane-binding site in a trigonal-planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Met residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport. Additionally, the studies indicate essential roles of other conserved intramembranous polar residues in facilitating copper binding to the high-affinity site and subsequent release through the exit pathway.