Two stacked heme molecules in the binding pocket of the periplasmic heme-binding protein HmuT from Yersinia pestis

The periplasmic binding protein HmuT from Yersinia pestis (YpHmuT) is a component of the heme uptake locus hmu and delivers bound hemin to the inner-membrane-localized, ATP-binding cassette (ABC) transporter HmuUV for translocation into the cytoplasm. The mechanism of this process, heme transport across the inner membrane of pathogenic bacteria, is currently insufficiently understood at the molecular level. Here we describe the crystal structures of the substrate-free and heme-bound states of YpHmuT, revealing two lobes with a central binding cleft. Superposition of the apo and holo states reveals a minor tilting motion of the lobes surrounding concomitant with heme binding. Unexpectedly, YpHmuT binds two stacked hemes in a central binding cleft that is larger than those of the homologous periplasmic heme-binding proteins ShuT and PhuT, both of which bind only one heme. The hemes bound to YpHmuT are coordinated via a tyrosine side chain that contacts the Fe atom of one heme and a histidine that contacts the Fe atom of the other heme. The coordinating histidine is only conserved in a subset of periplasmic heme binding proteins suggesting that its presence predicts the ability to bind two heme molecules simultaneously. The structural data are supported by spectroscopic binding studies performed in solution, where up to two hemes can bind to YpHmuT. Isothermal titration calorimetry suggests that the two hemes are bound in discrete, sequential steps and with dissociation constants (K_D) of ∼ 0.29  and ∼ 29 nM, which is similar to the affinities observed in other bacterial substrate binding proteins. Our findings suggest that the cognate ABC transporter HmuUV may simultaneously translocate two hemes per reaction cycle.     

Functional Characteristics of TauA Binding Protein from TauABC <Emphasis Type="Italic">Escherichia coli</Emphasis> System

Although TauA shares few common characteristics with other known periplasmic binding protein, TauA is a putative periplasmic binding protein, part of tauABCD gene cluster involved in sulfonate transport in sulphate starvation condition. This protein was expressed in E. coli BL 21 and purified before to assess its binding functionalities. Measurement of K _d value (mean 11.3 nM) by binding/dialysis studies revealed high affinity and specificity with taurine and also indicated that TauA possessed a unique binding site for its ligand. Comparisons with other periplasmic binding proteins suggests TauA plays a major role in ABC transport system and could be ideal candidate to serve as taurine catcher in biological fluids.     

The mechanism of nucleotide‐binding domain dimerization in the intact maltose transporter as studied by all‐atom molecular dynamics simulations

The Escherichia coli maltose transporter MalFGK₂‐E belongs to the protein superfamily of ATP‐binding cassette (ABC) transporters. This protein is composed of heterodimeric transmembrane domains (TMDs) MalF and MalG, and the homodimeric nucleotide‐binding domains (NBDs) MalK₂. In addition to the TMDs and NBDs, the periplasmic maltose binding protein MalE captures maltose and shuttle it to the transporter. In this study, we performed all‐atom molecular dynamics (MD) simulations on the maltose transporter and found that both the binding of MalE to the periplasmic side of the TMDs and binding of ATP to the MalK₂ are necessary to facilitate the conformational change from the inward‐facing state to the occluded state, in which MalK₂ is completely dimerized. MalE binding suppressed the fluctuation of the TMDs and MalF periplasmic region (MalF‐P2), and thus prevented the incorrect arrangement of the MalF C‐terminal (TM8) helix. Without MalE binding, the MalF TM8 helix showed a tendency to intrude into the substrate translocation pathway, hindering the closure of the MalK₂. This observation is consistent with previous mutagenesis experimental results on MalF and provides a new point of view regarding the understanding of the substrate translocation mechanism of the maltose transporter.     

A role for <Emphasis Type="Italic">Haemophilus ducreyi</Emphasis> Cu,ZnSOD in resistance to heme toxicity

The Cu,Zn superoxide dismutase (Cu,ZnSOD) from Haemophilus ducreyi is the only enzyme of this class which binds a heme molecule at its dimer interface. To explore the role of the enzyme in this heme-obligate bacterium, a sodC mutant was created by insertional inactivation. No difference in growth rate was observed during heme limitation. In contrast, under heme rich conditions growth of the sodC mutant was impaired compared to the wild type strain. This growth defect was abolished by supplementation of exogenous catalase. Genetic complementation of the sodC mutant in trans demonstrated that the enzymatic property or the heme-binding activity of the protein could repair the growth defect of the sodC mutant. These results indicate that Cu,ZnSOD protects Haemophilus ducreyi from heme toxicity.     

GxySBA ABC Transporter of Agrobacterium tumefaciens and Its Role in Sugar Utilization and vir Gene Expression

Monosaccharides available in the extracellular milieu of Agrobacterium tumefaciens can be transported into the cytoplasm, or via the periplasmic sugar binding protein, ChvE, play a critical role in controlling virulence gene expression. The ChvE-MmsAB ABC transporter is involved in the utilization of a wide range of monosaccharide substrates but redundant transporters are likely given the ability of a chvE-mmsAB deletion strain to grow, albeit more slowly, in the presence of particular monosaccharides. In this study, a putative ABC transporter encoded by the gxySBA operon is identified and shown to be involved in the utilization of glucose, xylose, fucose, and arabinose, which are also substrates for the ChvE-MmsAB ABC transporter. Significantly, GxySBA is also shown to be the first characterized glucosamine ABC transporter. The divergently transcribed gene gxyR encodes a repressor of the gxySBA operon, the function of which can be relieved by a subset of the transported sugars, including glucose, xylose, and glucosamine, and this substrate-induced expression can be repressed by glycerol. Furthermore, deletion of the transporter can increase the sensitivity of the virulence gene expression system to certain sugars that regulate it. Collectively, the results reveal a remarkably diverse set of substrates for the GxySBA transporter and its contribution to the repression of sugar sensitivity by the virulence-controlling system, thereby facilitating the capacity of the bacterium to distinguish between the soil and plant environments.     

Ferric hydroxamate binding protein FhuD from Escherichia coli: mutants in conserved and non-conserved regions

Uptake of iron complexes into the Gram-negative bacterial cell requires highly specific outer membrane receptors and specific ATP-dependent (ATP-Binding-Cassette (ABC)) transport systems located in the inner membrane. The latter type of import system is characterized by a periplasmic binding protein (BP), integral membrane proteins, and membrane-associated ATP-hydrolyzing proteins. In Gram-positive bacteria lacking the periplasmic space, the binding proteins are lipoproteins tethered to the cytoplasmic membrane. To date, there is little structural information about the components of ABC transport systems involved in iron complex transport. The recently determined structure of the Escherichia coli periplasmic ferric siderophore binding protein FhuD is unique for an ABC transport system (Clarke et al. 2000). Unlike other BP's, FhuD has two domains connected by a long -helix. The ligand binds in a shallow pocket between the two domains. In vivo and in vitro analysis of single amino acid mutants of FhuD identified several residues that are important for proper functioning of the protein. In this study, the mutated residues were mapped to the protein structure to define special areas and specific amino acid residues in E. coli FhuD that are vital for correct protein function. A number of these important residues were localized in conserved regions according to a multiple sequence alignment of E. coli FhuD with other BP's that transport siderophores, heme, and vitamin B₁₂. The alignment and structure prediction of these polypeptides indicate that they form a distinct family of periplasmic binding proteins.     

<Emphasis Type="Italic">Escherichia coli tat</Emphasis> mutant strains are able to transport maltose in the absence of an active <Emphasis Type="Italic">malE</Emphasis> gene

The twin-arginine transport (Tat) system is a prokaryotic protein transport system. Escherichia coli mutants in this pathway show a defect in cell separation during cell division, resulting in destabilization and permeability of the outer membrane. Maltose uptake is catalysed by a membrane-bound transporter of the ATP binding cassette (ABC) superfamily, where MalE is the essential periplasmic binding protein component. Here, we report that tat mutants are unexpectedly able to transport maltose in the absence of malE. This observation is specific to the MalE component since co-inactivation of malF, which encodes one of the channel components of the transporter, completely abolishes maltose transport even when the Tat system is inactivated. Genetic repair of the outer membrane leaky phenotype of the tat mutant strain re-established the absolute requirement for MalE in maltose uptake. In addition, we demonstrate that phenotypic repair of the outer membrane defect of the tat strain can also be achieved chemically by the inclusion of high concentrations of calcium or magnesium in the growth medium.     

Crystal structures of an Extracytoplasmic Solute Receptor from a TRAP transporter in its open and closed forms reveal a helix-swapped dimer requiring a cation for α-keto acid binding

Background  

The import of solutes into the bacterial cytoplasm involves several types of membrane transporters, which may be driven by ATP hydrolysis (ABC transporters) or by an ion or H⁺ electrochemical membrane potential, as in the tripartite ATP-independent periplasmic system (TRAP). In both the ABC and TRAP systems, a specific periplasmic protein from the ESR family (Extracytoplasmic Solute Receptors) is often involved for the recruitment of the solute and its presentation to the membrane complex. In Rhodobacter sphaeroides, TakP (previously named SmoM) is an ESR from a TRAP transporter and binds α-keto acids in vitro.     

Functional characterization of the Shigella dysenteriae heme ABC transporter

The heme ATP binding cassette (ABC) transporter, ShuUV, of Shigella dysenteriae has been incorporated into proteoliposomes. Functional characterization of ShuUV revealed that ATP hydrolysis and transport of heme from the periplasmic binding protein, ShuT, to the cytoplasmic binding protein, ShuS, are coupled. Site-directed mutagenesis of ShuT residues proposed to be required for stabilization of the complex abolished heme transport. Furthermore, residues His-252 and His-262, located in the translocation channel of ShuU, were required for the release of heme from ShuT and translocation to ShuS. The initial functional characterization of an in vitro heme uptake system provides a platform for future spectroscopic studies.     

A DING phosphatase in Thermus thermophilus

Summary. Phosphate transport in bacteria occurs via a phosphate specific transporter system (PSTS) that belongs to the ABC family of transporters, a multisubunit system, containing an alkaline phosphatase. DING proteins were characterized due to the N-terminal amino acid sequence DINGG GATL, which is highly conserved in animal and plant isolates, but more variable in microbes. Most prokaryotic homologues of the DING proteins often have some structural homology to phosphatases or periplasmic phosphate-binding proteins. In E. coli, the product of the inducible gene DinG, possesses ATP hydrolyzing helicase enzymic activity. An alkaline phosphorolytic enzyme of the PSTS system was purified to homogeneity from the thermophilic bacterium Thermus thermophilus. N-terminal sequence analysis of this protein revealed the same high degree of similarity to DING proteins especially to the human synovial stimulatory protein P205, the steroidogenesis-inducing protein and to the phosphate ABC transporter, periplasmic phosphate-binding protein, putative (P. fluorescens Pf-5). The enzyme had a molecular mass of 40 kDa on SDS/PAGE, exhibiting optimal phosphatase activity at pH 12.3 and 70 °C. The enzyme possessed characteristics of a DING protein, such as ATPase, ds endonuclease and 3′ phosphodiesterase (3′-exonuclease) activities and binding to linear dsDNA, displaying helicase activity on supercoiled DNA. Purification and biochemical characterization of a T. thermophilus DING protein was achieved. The biochemical properties, N-terminal sequence similarities of this protein implied that the enzyme belongs to the PSTS family and might be involved in the DNA repair mechanism of this microorganism. Authors’ address: Assist. Prof. A. A. Pantazaki, Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece     

Structural basis for binding and transfer of heme in bacterial heme‐acquisition systems

Periplasmic heme‐binding proteins (PBPs) in Gram‐negative bacteria are components of the heme acquisition system. These proteins shuttle heme across the periplasmic space from outer membrane receptors to ATP‐binding cassette (ABC) heme importers located in the inner‐membrane. In the present study, we characterized the structures of PBPs found in the pathogen Burkholderia cenocepacia (BhuT) and in the thermophile Roseiflexus sp. RS‐1 (RhuT) in the heme‐free and heme‐bound forms. The conserved motif, in which a well‐conserved Tyr interacts with the nearby Arg coordinates on heme iron, was observed in both PBPs. The heme was recognized by its surroundings in a variety of manners including hydrophobic interactions and hydrogen bonds, which was confirmed by isothermal titration calorimetry. Furthermore, this study of 3 forms of BhuT allowed the first structural comparison and showed that the heme‐binding cleft of BhuT adopts an “open” state in the heme‐free and 2‐heme‐bound forms, and a “closed” state in the one‐heme‐bound form with unique conformational changes. Such a conformational change might adjust the interaction of the heme(s) with the residues in PBP and facilitate the transfer of the heme into the translocation channel of the importer.     

Involvement of Agrobacterium tumefaciens Galacturonate Tripartite ATP-Independent Periplasmic (TRAP) Transporter GaaPQM in Virulence Gene Expression

Monosaccharides capable of serving as nutrients for the soil bacterium Agrobacterium tumefaciens are also inducers of the vir regulon present in the tumor-inducing (Ti) plasmid of this plant pathogen. One such monosaccharide is galacturonate, the predominant monomer of pectin found in plant cell walls. This ligand is recognized by the periplasmic sugar binding protein ChvE, which interacts with the VirA histidine kinase that controls vir gene expression. Although ChvE is also a member of the ChvE-MmsAB ABC transporter involved in the utilization of many neutral sugars, it is not involved in galacturonate utilization. In this study, a putative tripartite ATP-independent periplasmic (TRAP) transporter, GaaPQM, is shown to be essential for the utilization of galacturonic acid; we show that residue R169 in the predicted sugar binding site of the GaaP is required for activity. The gene upstream of gaaPQM (gaaR) encodes a member of the GntR family of regulators. GaaR is shown to repress the expression of gaaPQM, and the repression is relieved in the presence of the substrate for GaaPQM. Moreover, GaaR is shown to bind putative promoter regions in the sequences required for galacturonic acid utilization. Finally, A. tumefaciens strains carrying a deletion of gaaPQM are more sensitive to galacturonate as an inducer of vir gene expression, while the overexpression of gaaPQM results in strains being less sensitive to this vir inducer. This supports a model in which transporter activity is crucial in ensuring that vir gene expression occurs only at sites of high ligand concentration, such as those at a plant wound site.     

Full engagement of liganded maltose‐binding protein stabilizes a semi‐open ATP‐binding cassette dimer in the maltose transporter

MalFGK₂ is an ATP‐binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose‐binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide‐binding subunits (MalK dimer). This MBP‐stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose‐bound MBP unto MalFGK₂ is facilitated by nucleotides and stabilizes a semi‐open MalK dimer. Maltose‐bound MBP promotes the transition to the semi‐open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi‐open MalK₂ conformation by maltose‐bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi‐open conformation, from which it can proceed to hydrolyze ATP.     

Detection and characterization of an ABC transporter in <Emphasis Type="Italic">Clostridium hathewayi</Emphasis>

An ABC transporter gene from Clostridium hathewayi is characterized. It has duplicated ATPase domains in addition to a transmembrane protein. Its deduced amino acid sequence has conserved functional domains with ATPase components of the multidrug efflux pump genes of several bacteria. Cloning this transporter gene into C. perfringens and E. coli resulted in decreased sensitivities of these bacteria to fluoroquinolones. It also decreased the accumulation and increased the efflux of ethidium bromide from cells containing the cloned gene. Carbonyl cyanide-m-chlorophenylhydrazone (CCCP) inhibited both accumulation and efflux of ethidium bromide from these cells. The ATPase mRNA was overexpressed in the fluoroquinolone-resistant strain when exposed to ciprofloxacin. This is the first report of an ABC transporter in C. hathewayi. An erratum to this article can be found at     

Structural similarities between thiamin-binding protein and thiaminase-I suggest a common ancestor

ATP-binding cassette (ABC) transporters are responsible for the transport of a wide variety of water-soluble molecules and ions into prokaryotic cells. In Gram-negative bacteria, periplasmic-binding proteins deliver ions or molecules such as thiamin to the membrane-bound ABC transporter. The gene for the thiamin-binding protein tbpA has been identified in both Escherichia coli and Salmonella typhimurium. Here we report the crystal structure of TbpA from E. coli with bound thiamin monophosphate. The structure was determined at 2.25 A resolution using single-wavelength anomalous diffraction experiments, despite the presence of nonmerohedral twinning. The crystal structure shows that TbpA belongs to the group II periplasmic-binding protein family. Equilibrium binding measurements showed similar dissociation constants for thiamin, thiamin monophosphate, and thiamin pyrophosphate. Analysis of the binding site by molecular modeling demonstrated how TbpA binds all three forms of thiamin. A comparison of TbpA and thiaminase-I, a thiamin-degrading enzyme, revealed structural similarity between the two proteins, especially in domain 1, suggesting that the two proteins evolved from a common ancestor.     

NikA binds heme: a new role for an Escherichia coli periplasmic nickel-binding protein

NikA is a periplasmic binding protein involved in nickel uptake in Escherichia coli. NikA was identified as a heme-binding protein in the periplasm of anaerobically grown cells overexpressing CydDC, an ABC transporter that exports reductant to the periplasm. CydDC-overexpressing cells accumulate a heme biosynthesis-derived pigment, P-574. For further biochemical and spectroscopic analysis, unliganded NikA was overexpressed and purified. NikA was found to comigrate with both hemin and protoporphyrin IX during gel filtration. Furthermore, tryptophan fluorescence quenching titrations demonstrated that both hemin and protoporphyrin IX bind to NikA with similar affinity. The binding affinity of NikA for these pigments (Kd approximately 0.5 microM) was unaltered in the presence and absence of saturating concentrations of nickel, suggesting that these tetrapyrroles bind to NikA in a manner independent of nickel. To test the hypothesis that NikA is required for periplasmic heme protein assembly, the effects of a nikA mutation (nikA::Tn5, Km(R) insertion) on accumulation of P-574 by CydDC-overexpressing cells was assessed. This mutation significantly lowered P-574 levels, implying that NikA may be involved in P-574 production. Thus, in the reducing environment of the periplasm, NikA may serve as a heme chaperone as well as a periplasmic nickel-binding protein. The docking of heme onto NikA was modeled using the published crystal structure; many of the predicted complexes exhibit a heme-binding cleft remote from the nickel-binding site, which is consistent with the independent binding of nickel and heme. This work has implications for the incorporation of heme into b- and c-type cytochromes.     

Proteomics analysis reveals that CirA in Aeromonas hydrophila is involved in nutrient uptake

The colicin I receptor (CirA) is a well-studied outer membrane protein that has been reported to play important roles in antibiotic resistance, virulence, and iron homeostasis, although its exact physiological roles require further investigation. In this study, differentially expressed proteins between the ΔahcirA and wild-type (WT) strains of Aeromonas hydrophila were compared using quantitative proteomics. Bioinformatics analysis revealed that the expression of peptide, histidine, and arginine ATP-binding cassette (ABC) transporter system-related proteins was significantly higher in the ΔahcirA strain. Subsequent growth assays revealed that ΔahcirA grew slower than the WT strain in nutrient-limited medium when supplemented with dipeptide, histidine, and arginine as the carbon source. Far-western blot analysis further confirmed that AhCirA can directly bind to histidine/arginine and dipeptide small-molecule substrates in addition to their periplasmic-binding proteins, AhDppA and AhHisJ, respectively. These results indicate that AhCirA may play an important role in the uptake of amino acids and peptides as a channel-forming porin while also directly interacting with ABC transporters to transport nutrient substances into the plasma membrane. Overall, this study demonstrates that AhCirA is a multifunctional protein in A. hydrophila and extends our understanding of known nutrient transport mechanisms among bacteria.     

Structures of the Neisseria meningitides methionine‐binding protein MetQ in substrate‐free form and bound to l‐ and d‐methionine isomers

The bacterial periplasmic methionine‐binding protein MetQ is involved in the import of methionine by the cognate MetNI methionine ATP binding cassette (ABC) transporter. The MetNIQ system is one of the few members of the ABC importer family that has been structurally characterized in multiple conformational states. Critical missing elements in the structural analysis of MetNIQ are the structure of the substrate‐free form of MetQ, and detailing how MetQ binds multiple methionine derivatives, including both l ‐ and d ‐methionine isomers. In this study, we report the structures of the Neisseria meningitides MetQ in substrate‐free form and in complexes with l ‐methionine and with d ‐methionine, along with the associated binding constants determined by isothermal titration calorimetry. Structures of the substrate‐free (N238A) and substrate‐bound N. meningitides MetQ are related by a “Venus‐fly trap” hinge‐type movement of the two domains accompanying methionine binding and dissociation. l ‐ and d ‐methionine bind to the same site on MetQ, and this study emphasizes the important role of asparagine 238 in ligand binding and affinity. A thermodynamic analysis demonstrates that ligand‐free MetQ associates with the ATP‐bound form of MetNI ～40 times more tightly than does liganded MetQ, consistent with the necessity of dissociating methionine from MetQ for transport to occur.