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1.  Structural insights into the transport of small molecules across membranes 
While hydrophobic small molecules often can freely permeate a lipid bilayer, ions and other polar molecules cannot and require transporters to mediate their transport. Recently, a number of important structures have been reported which have advanced our understanding of how membrane protein transporters function to transport small molecules. Structures of TbpA/B and HmuUV provided new insight into iron uptake by pathogenic bacteria while the structures of NarK, ASBT, and VcINDY revealed molecular details about the transport of nitrate, bile acids and dicarboxylates, respectively. The structure of the folate ECF transporter indicated that the S component likely undergoes a large conformational shift to mediate folate transport, while the cellulose synthase/transporter contains an elongated translocation pore for passage through the inner membrane.
PMCID: PMC4177025  PMID: 24681594
2.  Lateral opening and exit pore formation are required for BamA function 
Structure (London, England : 1993)  2014;22(7):1055-1062.
The outer membrane (OM) of Gram-negative bacteria is replete with a host of β-barrel outer membrane proteins (OMPs). Despite serving a variety of essential functions, including immune response evasion, the exact mechanism of OMP folding and membrane insertion remains largely unclear. The β-barrel assembly machinery (BAM) complex is required for OMP biogenesis. Crystal structures and molecular dynamics (MD) simulations of the central and essential component, BamA, suggest a mechanism involving lateral opening of its barrel domain. MD simulations reported here reveal an additional feature of BamA: a substrate exit pore positioned above the lateral opening site. Disulfide crosslinks that prevent lateral opening and exit pore formation result in a loss of BamA function, which can be fully rescued by the reductant TCEP. These data provide strong evidence that lateral opening and exit pore formation are required for BamA function.
PMCID: PMC4100585  PMID: 24980798
3.  FhaC takes a bow to FHA in the two-partner do-si-do 
Molecular microbiology  2014;92(6):1155-1158.
FhaC is an outer membrane transporter from Bordetella pertussis belonging to the two-partner secretion (TPS) pathway with its primary role being the secretion of the virulence factor filamentous haemagglutinin (FHA). FhaC serves as a model transporter of the TPS pathway and significant work has been done to characterize the role of FhaC in FHA secretion. Recent studies characterized interactions between FHA and the POTRA domains of FhaC, suggesting that secretion may involve a successive translocation mechanism mediated by β-augmentation and/or electrostatic interactions. Moreover, it was also shown that reconstituted FhaC is necessary and sufficient to transport FHA into proteoliposomes. While the crystal structure of FhaC clearly suggests a role in transport, the putative transport pore is plugged by an N-terminal α-helix (H1 helix) that occludes access by FHA. Therefore, it has been proposed that the H1 helix must be expelled from the pore in order for secretion of FHA to occur. However, this has yet to be shown experimentally. In this issue of Molecular Microbiology, Guérin et al. report the first direct experimental evidence to show that the FhaC H1 helix is quite dynamic and exchanges between closed and open states upon interaction with FHA.
PMCID: PMC4051853  PMID: 24798489
4.  Molecular Insight into Substrate Recognition and Catalysis of Baeyer–Villiger Monooxygenase MtmOIV, the Key Frame Modifying Enzyme in the Biosynthesis of Anticancer Agent Mithramycin 
ACS chemical biology  2013;8(11):10.1021/cb400399b.
Baeyer-Villiger monooxygenases (BVMOs) have been shown to play key roles for the biosynthesis of important natural products. MtmOIV, a homodimeric FAD- and NADPH-dependent BVMO, catalyzes the key frame-modifying steps of the mithramycin biosynthetic pathway, including an oxidative C-C bond cleavage, by converting its natural substrate premithramycin B into mithramycin DK, the immediate precursor of mithramycin. The drastically improved protein structure of MtmOIV along with the high-resolution structure of MtmOIV in complex with its natural substrate premithramycin B are reported here, revealing previously undetected key residues that are important for substrate recognition and catalysis. Kinetic analyses of selected mutants allowed us to probe the substrate binding pocket of MtmOIV, and also to discover the putative NADPH binding site. This is the first substrate-bound structure of MtmOIV providing new insights into substrate recognition and catalysis, which paves the way for the future design of a tailored enzyme for the chemo-enzymatic preparation of novel mithramycin analogues.
PMCID: PMC3830731  PMID: 23992662
The Journal of biological chemistry  2006;281(34):24934-24944.
Serum transferrin reversibly binds iron in each of two lobes and delivers it to cells by a receptor-mediated, pH-dependant process. The binding and release of iron results in a large conformational change in which two subdomains in each lobe close or open with a rigid twisting motion around a hinge. We report the structure of human serum transferrin (hTF) lacking iron (apo-hTF) which was independently determined by two methods: (1) the crystal structure of recombinant non-glycosylated apo-hTF was solved at 2.7 Å resolution using a MAD phasing strategy, by substituting the nine methionines in hTF with selenomethionine and (2) the structure of glycosylated apo-hTF (isolated from serum) was determined to a resolution of 2.7 Å by molecular replacement using the human apo-N-lobe and the rabbit holo-C1-subdomain as search models. These two crystal structures are essentially identical. They represent the first published model for full-length human TF and reveal that, in contrast to family members (human lactoferrin and hen ovotransferrin), both lobes are almost equally open: 59.4° and 49.5° rotations are required to open the N- and C-lobe, respectively, (compared to closed pig TF). Availability of this structure is critical to a complete understanding of the metal binding properties of each lobe of hTF; the apo-hTF structure suggests that differences in the hinge regions of the N- and C-lobes may influence the rates of iron binding and release. In addition, we evaluate potential interactions between apo-hTF and the human transferrin receptor.
PMCID: PMC1895924  PMID: 16793765
6.  Structural insight into the lactoferrin receptors from pathogenic Neisseria 
Journal of structural biology  2013;184(1):83-92.
Neisseria are pathogenic bacteria that cause gonorrhea, septicemia, and meningitis. Like other pathogenic bacteria, Neisseria must acquire iron for survival from their local environment within the human host. Instead of secreting siderophores to scavenge iron, Neisseria steal iron from human iron binding proteins such as hemoglobin, transferrin and lactoferrin for survival. Recently we reported the crystal structures of the N. meningitidis transferrin receptors TbpA and TbpB, as well as the structures of apo and holo human transferrin. We also analyzed these proteins using small angle X-ray scattering and electron microscopy to provide the molecular details explaining how Neisseria are able to interact with and extract iron from transferrin. Here, we utilize the structural reports, as well as the recently reported structure of the N-lobe of LbpB from Moraxella bovis, to assemble improved 3D homology models for the neisserial lactoferrin import receptors LbpA and LbpB, both of which are important vaccine targets against N. meningitidis. We then analyzed these models to gain structural insights into the lactoferrin-iron import system and form a mechanistic model fashioned in parallel to the homologous transferrin-iron import system.
PMCID: PMC3716850  PMID: 23462098
Neisseria; meningitidis; gonorrhoeae; lactoferrin; transferrin; iron acquisition; TonB
7.  An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins 
PLoS ONE  2014;9(4):e95808.
Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu2+, Ni2+, Co2+, and Zn2+). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.
PMCID: PMC3994163  PMID: 24752441
8.  Structural insight into the biogenesis of β-barrel membrane proteins 
Nature  2013;501(7467):385-390.
β-barrel membrane proteins are essential for nutrient import, signaling, motility, and survival. In Gram-negative bacteria, the β-barrel assembly machinery (BAM) complex is responsible for the biogenesis of β-barrel membrane proteins, with homologous complexes found in mitochondria and chloroplasts. Here we describe the structure of BamA, the central and essential component of the BAM complex, from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane β-barrel domain. Three structural features speak to the mechanism by which BamA catalyzes β-barrel assembly. First, the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the β-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. And third, the β-barrel can undergo lateral opening, evocatively suggesting a route from the interior cavity in BamA into the outer membrane.
PMCID: PMC3779476  PMID: 23995689
9.  The transferrin-iron import system from pathogenic Neisseria species 
Molecular microbiology  2012;86(2):246-257.
The two pathogenic species within the genus Neisseria cause the diseases gonorrhea and meningitis. While vaccines are available to protect against four N. meningitidis serogroups, there is currently no commercial vaccine to protect against serogroup B or against N. gonorrhoeae. Moreover, the available vaccines have significant limitations and with antibiotic resistance becoming an alarming issue, the search for effective vaccine targets to elicit long-lasting protection against Neisseria species is becoming more urgent. One strategy for vaccine development has targeted the neisserial iron import systems. Without iron, the Neisseriae cannot survive and therefore these iron import systems tend to be relatively well conserved and are promising vaccine targets, having the potential to offer broad protection against both gonococcal and meningococcal infections. These efforts have been boosted by recent reports of the crystal structures of the neisserial receptor proteins TbpA and TbpB, each solved in complex with human transferrin, an iron binding protein normally responsible for delivering iron to human cells. Here, we review the recent structural reports and put them into perspective with available functional studies in order to derive the mechanism(s) for how the pathogenic Neisseriae are able to hijack human iron transport systems for their own survival and pathogenesis.
PMCID: PMC3468669  PMID: 22957710
10.  Dynamic association of BAM complex modules includes surface exposure of the lipoprotein BamC. 
Journal of molecular biology  2012;422(4):545-555.
The BAM complex drives assembly of β-barrel proteins into the outer membrane of gram-negative bacteria. It is composed of five subunits: BamA, BamB, BamC, BamD and BamE. We find that the BAM complex isolated from the outer membrane of Escherichia coli consists of a core complex of BamA:B:C:D:E and in addition, a BamA:B module and a BamC:D module. In the absence of BamC, these modules are destabilized resulting in increased protease susceptibility of BamD and BamB. While the N-terminus of BamC carries a highly conserved region crucial for stable interaction with BamD, immunofluorescence, immunoprecipitation and protease-sensitivity assays show that the C-terminal domain of BamC, comprised of two helix-grip motifs, is exposed on the surface of Escherichia coli. This unexpected topology of a bacterial lipoprotein is reminiscent of the analogous protein subunits from the mitochondrial β-barrel insertion machinery, the SAM complex. The modular arrangement and topological features provide new insight into the architecture of the BAM complex, towards a better understanding of the mechanism driving β-barrel membrane protein assembly.
PMCID: PMC3433275  PMID: 22683355
β-barrel proteins; Omp85; outer membrane biogenesis; protein transport
11.  Crystal Structures of the Outer Membrane Domain of Intimin and Invasin from Enterohemorrhagic Escherichia coli and Enteropathogenic Yersinia pseudotuberculosis 
Structure(London, England:1993)  2012;20(7):1233-1243.
Intimins and invasins are virulence factors produced by pathogenic Gram-negative bacteria. They contain C-terminal extracellular passenger domains that are involved in adhesion to host cells and N-terminal β-domains that are embedded in the outer membrane. Here, we identify the domain boundaries of an E. coli intimin β-domain and use this information to solve its structure and the β-domain structure of a Y. pseudotuberculosis invasin. Both β-domain structures crystallized as monomers and reveal that the previous range of residues assigned to the β-domain also includes a protease resistant domain that is part of the passenger. Additionally, we identify 146 non-redundant representative members of the intimin/invasin family based on the boundaries of the highly conserved intimin and invasin β-domains. We then use this set of sequences along with our structural data to find and map the evolutionarily constrained residues within the β-domain.
PMCID: PMC3392549  PMID: 22658748
12.  Using a bacteriocin structure to engineer a phage lysin that targets Yersinia pestis 
Biochemical Society transactions  2012;40(6):1503-1506.
Purified phage lysins present an alternative to traditional antibiotics and work by hydrolyzing peptidoglycan. Phage lysins have been developed against Gram-positive pathogens such as Bacillus anthracis and Streptococcus pneumoniae, where the peptidoglycan layer is exposed on the cell surface. Addition of the lysin to a bacterial culture results in rapid death of the organism. Gram-negative bacteria are resistant to phage lysins because they contain an outer membrane that protects the peptidoglycan from degradation. We solved crystal structures of a Yersinia pestis outer membrane protein and the bacteriocin that targets it, which informed engineering of a bacterial-phage hybrid lysin that can be transported across the outer membrane to kill specific Gram-negative bacteria. This work provides a template for engineering phage lysins against a wide variety of bacterial pathogens.
PMCID: PMC3679646  PMID: 23176506
13.  Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline 
Journal of Synchrotron Radiation  2013;20(Pt 4):531-540.
Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction for combined single-platform analysis, examining the viability of NLO microscopy as an alternative to the conventional X-ray raster scan for the purposes of sample centering. Second-harmonic generation microscopy and two-photon excited ultraviolet fluorescence microscopy were evaluated for crystal detection, and assessed by X-ray raster scanning.
Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼103–104-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied.
PMCID: PMC3682636  PMID: 23765294
XRD; NLO; SHG; SONICC; centering; protein; TPE-UVF; microscopy; LCP; two-photon
14.  Molecular Basis for Activation of a Catalytic Asparagine Residue in a Self-Cleaving Bacterial Autotransporter 
Journal of molecular biology  2011;415(1):128-142.
Autotransporters are secreted proteins produced by pathogenic Gram-negative bacteria. They consist of a membrane embedded β-domain and an extracellular passenger domain that is sometimes cleaved and released from the cell surface. We solved the structures of three non-cleavable mutants of the autotransporter EspP to examine how it promotes asparagine cyclization to cleave its passenger. We found that cyclization is facilitated by multiple factors. The active site asparagine is sterically constrained to conformations favorable for cyclization while electrostatic interactions correctly orient the carboxamide group for nucleophilic attack. During molecular dynamics simulations, water molecules were observed to enter the active site and form hydrogen bonds favorable for increasing the nucleophilicity of the active site asparagine. When the activated asparagine attacks its main chain carbonyl carbon the resulting oxyanion is stabilized by a protonated glutamate. Upon cleavage, this proton could be transferred to the leaving amine group helping overcome a significant energy barrier. Together these findings provide insight into factors important for asparagine cyclization, a broadly used mechanism for protein cleavage.
PMCID: PMC3230255  PMID: 22094314
EspP; autocleavage; outer membrane protein; crystal structure; asparagine cyclization
15.  Specific targeting and killing of Gram-negative pathogens with an engineered phage lytic enzyme 
Virulence  2013;4(1):90-91.
PMCID: PMC3544754  PMID: 23314572
pesticin; FyuA; plague; phage
16.  Structural insights into Ail-mediated adhesion in Yersinia pestis 
Structure (London, England : 1993)  2011;19(11):1672-1682.
Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin, and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells.
PMCID: PMC3217190  PMID: 22078566
ail; plague; Yersinia pestis; adhesion; invasion; extracellular matrix proteins; outer membrane protein; crystal structure
17.  The structural biology of β-barrel membrane proteins: a summary of recent reports 
The outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts all contain transmembrane β-barrel proteins. These β-barrel proteins serve essential functions in cargo transport and signaling and are also vital for membrane biogenesis. They have also been adapted to perform a diverse set of important cellular functions including acting as porins, transporters, enzymes, virulence factors and receptors. Recent structures of transmembrane β-barrels include that of a full length autotransporter (EstA), a bacterial heme transporter complex (HasR), a bacterial porin in complex with several ligands (PorB), and the mitochondrial voltage-dependent anion channel (VDAC) from both mouse and human. These represent only a few of the interesting structures of β-barrel membrane proteins recently elucidated. However, they demonstrate many of the advancements made within the field of transmembrane protein structure in the past few years.
PMCID: PMC3164749  PMID: 21719274
18.  Structural Insights into the Catalytic Mechanism of Escherichia coli Selenophosphate Synthetase 
Journal of Bacteriology  2012;194(2):499-508.
Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. SPS activity is dependent on both Mg2+ and K+ and uses ATP, selenide, and water to catalyze the formation of AMP, orthophosphate, and selenophosphate. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Most of what is known about the function of SPS has derived from studies investigating Escherichia coli SPS (EcSPS) as a model system. Here we report the crystal structure of the C17S mutant of SPS from E. coli (EcSPSC17S) in apo form (without ATP bound). EcSPSC17S crystallizes as a homodimer, which was further characterized by analytical ultracentrifugation experiments. The glycine-rich N-terminal region (residues 1 through 47) was found in the open conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished AMP production in our activity assays, highlighting their essential role for catalysis in EcSPS. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies done with SPS orthologs from Aquifex aeolicus and humans, we propose a catalytic mechanism for EcSPS-mediated selenophosphate synthesis.
PMCID: PMC3256651  PMID: 22081394
19.  Role of the Yersinia pestis Ail Protein in Preventing a Protective Polymorphonuclear Leukocyte Response during Bubonic Plague▿  
Infection and Immunity  2011;79(12):4984-4989.
The ability of Yersinia pestis to forestall the mammalian innate immune response is a fundamental aspect of plague pathogenesis. In this study, we examined the effect of Ail, a 17-kDa outer membrane protein that protects Y. pestis against complement-mediated lysis, on bubonic plague pathogenesis in mice and rats. The Y. pestis ail mutant was attenuated for virulence in both rodent models. The attenuation was greater in rats than in mice, which correlates with the ability of normal rat serum, but not mouse serum, to kill ail-negative Y. pestis in vitro. Intradermal infection with the ail mutant resulted in an atypical, subacute form of bubonic plague associated with extensive recruitment of polymorphonuclear leukocytes (PMN or neutrophils) to the site of infection in the draining lymph node and the formation of large purulent abscesses that contained the bacteria. Systemic spread and mortality were greatly attenuated, however, and a productive adaptive immune response was generated after high-dose challenge, as evidenced by high serum antibody levels against Y. pestis F1 antigen. The Y. pestis Ail protein is an important bubonic plague virulence factor that inhibits the innate immune response, in particular the recruitment of a protective PMN response to the infected lymph node.
PMCID: PMC3232667  PMID: 21969002
20.  Ancestral and Derived Protein Import Pathways in the Mitochondrion of Reclinomonas americana 
Molecular Biology and Evolution  2010;28(5):1581-1591.
The evolution of mitochondria from ancestral bacteria required that new protein transport machinery be established. Recent controversy over the evolution of these new molecular machines hinges on the degree to which ancestral bacterial transporters contributed during the establishment of the new protein import pathway. Reclinomonas americana is a unicellular eukaryote with the most gene-rich mitochondrial genome known, and the large collection of membrane proteins encoded on the mitochondrial genome of R. americana includes a bacterial-type SecY protein transporter. Analysis of expressed sequence tags shows R. americana also has components of a mitochondrial protein translocase or “translocase in the inner mitochondrial membrane complex.” Along with several other membrane proteins encoded on the mitochondrial genome Cox11, an assembly factor for cytochrome c oxidase retains sequence features suggesting that it is assembled by the SecY complex in R. americana. Despite this, protein import studies show that the RaCox11 protein is suited for import into mitochondria and functional complementation if the gene is transferred into the nucleus of yeast. Reclinomonas americana provides direct evidence that bacterial protein transport pathways were retained, alongside the evolving mitochondrial protein import machinery, shedding new light on the process of mitochondrial evolution.
PMCID: PMC3080133  PMID: 21081480
mitochondria; protein import; Reclinomonas americana; transport pathway; translocon; SecY
21.  The crystal structure of BamB suggests interactions with BamA and its role within the BAM complex 
Journal of molecular biology  2011;407(2):248-260.
E. coli BamB is the largest of four lipoproteins in the β-barrel assembly machinery (BAM) complex. It interacts with the periplasmic domain of BamA, an integral outer membrane protein essential for outer membrane protein biogenesis. Although BamB is not essential, it serves an important function in the BAM complex, significantly increasing the folding efficiency of some OMPs in vivo and in vitro. To learn more about the BAM complex, we solved structures of BamB in three different crystal forms. BamB crystallized in space groups P213, I222, and P212121, with one molecule per asymmetric unit in each case. Crystals from the space group I222 diffracted to 1.65 Å resolution. BamB forms an 8-bladed β-propeller with a central pore and is shaped like a doughnut. A DALI search revealed that BamB shares structural homology to several eukaryotic proteins containing WD40 repeat domains, which commonly have β-propeller folds and often serve as scaffolding proteins within larger multi-protein complexes that carry out signal transduction, cell division, and chemotaxis. Using mutagenesis data from previous studies, we docked BamB onto a BamA structural model and assessed known and possible interactions between these two proteins. Our data suggest that BamB serves as a scaffolding protein within the BAM complex by optimally orienting the flexible periplasmic domain of BamA for interaction with other BAM components and chaperones. This may facilitate integration of newly synthesized outer membrane proteins into the outer membrane.
PMCID: PMC3048904  PMID: 21277859
22.  TonB-dependent transporters: regulation, structure, and function 
TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that bind and transport ferric chelates called siderophores, as well as vitamin B12, nickel complexes, and carbohydrates. The transport process requires energy in the form of protonmotive force and a complex of three inner membrane proteins, TonB-ExbB-ExbD, to transduce this energy to the outer membrane. The siderophore substrates range in complexity from simple small molecules such as citrate to large proteins like serum transferrin and haemoglobin. Because iron uptake is vital for almost all bacteria, expression of TBDTs is regulated in a number of ways that include metal-dependent regulators, σ/anti-σ factor systems, small RNAs, and even a riboswitch. In recent years many new structures of TBDTs have been solved in various states, resulting in a more complete picture of siderophore selectivity and binding, signal transduction across the outer membrane, and interaction with TonB-ExbB-ExbD. However, the transport mechanism is still unclear. In this review, we summarize recent progress in understanding regulation, structure and function in TBDTs and questions remaining to be answered.
PMCID: PMC3108441  PMID: 20420522
TonB-dependent transporter; outer membrane protein; siderophore; active transport; signal transduction
23.  The Protein Import Channel in the Outer Mitosomal Membrane of Giardia intestinalis 
Molecular Biology and Evolution  2009;26(9):1941-1947.
The identification of mitosomes in Giardia generated significant debate on the evolutionary origin of these organelles, whether they were highly reduced mitochondria or the product of a unique endosymbiotic event in an amitochondrial organism. As the protein import pathway is a defining characteristic of mitochondria, we sought to discover a TOM (translocase in the outer mitochondrial membrane) complex in Giardia. A Hidden Markov model search of the Giardia genome identified a Tom40 homologous sequence (GiTom40), where Tom40 is the protein translocation channel of the TOM complex. The GiTom40 protein is located in the membrane of mitosomes in a ∼200-kDa TOM complex. As Tom40 was derived in the development of mitochondria to serve as the protein import channel in the outer membrane, its presence in Giardia evidences the mitochondrial ancestry of mitosomes.
PMCID: PMC2734158  PMID: 19531743
mitochondria; mitosomes; Giardia intestinalis; protein translocation; evolution
24.  A structural comparison of human serum transferrin and human lactoferrin 
Biometals  2007;20(3-4):249-262.
The transferrins are a family of proteins that bind free iron in the blood and bodily fluids. Serum transferrins function to deliver iron to cells via a receptor-mediated endocytotic process as well as to remove toxic free iron from the blood and to provide an anti-bacterial, low-iron environment. Lactoferrins (found in bodily secretions such as milk) are only known to have an anti-bacterial function, via their ability to tightly bind free iron even at low pH, and have no known transport function. Though these proteins keep the level of free iron low, pathogenic bacteria are able to thrive by obtaining iron from their host via expression of outer membrane proteins that can bind to and remove iron from host proteins, including both serum transferrin and lactoferrin. Furthermore, even though human serum transferrin and lactoferrin are quite similar in sequence and structure, and coordinate iron in the same manner, they differ in their affinities for iron as well as their receptor binding properties: the human transferrin receptor only binds serum transferrin, and two distinct bacterial transport systems are used to capture iron from serum transferrin and lactoferrin. Comparison of the recently solved crystal structure of iron-free human serum transferrin to that of human lactoferrin provides insight into these differences.
PMCID: PMC2547852  PMID: 17216400
Transferrin; Lactoferrin; Iron transport; Human transferrin receptor; Bacterial transferrin receptor
25.  Signaling Mechanisms for Activation of Extracytoplasmic Function (ECF) Sigma Factors 
Biochimica et biophysica acta  2007;1778(9):1930-1945.
A variety of mechanisms are used to signal extracytoplasmic conditions to the cytoplasm. These mechanisms activate extracytoplasmic function (ECF) sigma factors which recruit RNA-polymerase to specific genes in order to express appropriate proteins in response to the changing environment. The two best understood ECF signaling pathways regulate σE-mediated expression of periplasmic stress response genes in Escherichia coli and FecI-mediated expression of iron-citrate transport genes in E. coli. Homologues from other Gram-negative bacteria suggest that these two signaling mechanisms and variations on these mechanisms may be the general schemes by which ECF sigma factors are regulated in Gram-negative bacteria.
PMCID: PMC2562455  PMID: 17673165
ECF; sigma factor; anti-sigma factor; sigma E; FecI

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