Steric and allosteric factors prevent simultaneous binding of transferrin-binding proteins A and B to transferrin

2012 ◽  
Vol 444 (2) ◽  
pp. 189-197 ◽  
Author(s):  
Leslie P. Silva ◽  
Rong-hua Yu ◽  
Charles Calmettes ◽  
Xue Yang ◽  
Trevor F. Moraes ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Protein ◽  
Iron Acquisition ◽  
Protein A ◽  
Structural Instability ◽  
Gated Channel ◽  
Surface Receptor ◽  
Hydrogen Deuterium ◽  
Study Support ◽  
Transferrin Binding Proteins

The ability to acquire iron directly from host Tf (transferrin) is an adaptation common to important bacterial pathogens belonging to the Pasteurellaceae, Moraxellaceae and Neisseriaceae families. A surface receptor comprising an integral outer membrane protein, TbpA (Tf-binding protein A), and a surface-exposed lipoprotein, TbpB (Tf-binding protein B), mediates the iron acquisition process. TbpB is thought to extend from the cell surface for capture of Tf to initiate the process and deliver Tf to TbpA. TbpA functions as a gated channel for the passage of iron into the periplasm. In the present study we have mapped the effect of TbpA from Actinobacillus pleuropneumoniae on pTf (porcine Tf) using H/DX-MS (hydrogen/deuterium exchange coupled to MS) and compare it with a previously determined binding site for TbpB. The proposed TbpA footprint is adjacent to and potentially overlapping the TbpB-binding site, and induces a structural instability in the TbpB site. This suggests that simultaneous binding to pTf by both receptors would be hindered. We demonstrate that a recombinant TbpB lacking a portion of its anchor peptide is unable to form a stable ternary TbpA–pTf–TbpB complex. This truncated TbpB does not bind to a preformed Tf–TbpA complex, and TbpA removes pTf from a preformed Tf–TbpB complex. Thus the results of the present study support a model whereby TbpB ‘hands-off’ pTf to TbpA, which completes the iron removal and transport process.

Crystal structure of the N-lobe of lactoferrin binding protein B from Moraxella bovis1 1This paper is an invited article as a result of a presentation at the International Lactoferrin Conference held in Mazatlan, Mexico (May 2011), and has undergone the Journal’s usual peer review process.

2012 ◽  
Vol 90 (3) ◽  
pp. 351-361 ◽  
Author(s):  
Elena Arutyunova ◽  
Cory L. Brooks ◽  
Amanda Beddek ◽  
Michelle W. Mak ◽  
Anthony B. Schryvers ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Information ◽  
Peer Review Process ◽  
Binding Protein ◽  
Iron Acquisition ◽  
Protein A ◽  
Bacterial Surface ◽  
Moraxella Bovis ◽  
Surface Receptor ◽  
Protein B

Lactoferrin (Lf) is a bi-lobed, iron-binding protein found on mucosal surfaces and at sites of inflammation. Gram-negative pathogens from the Neisseriaceae and Moraxellaceae families are capable of using Lf as a source of iron for growth through a process mediated by a bacterial surface receptor that directly binds host Lf. This receptor consists of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a surface lipoprotein, lactoferrin binding protein B (LbpB). The N-lobe of the homologous transferrin binding protein B, TbpB, has been shown to facilitate transferrin binding in the process of iron acquisition. Currently there is little known about the role of LbpB in iron acquisition or how Lf interacts with the bacterial receptor proteins. No structural information on any LbpB or domain is available. In this study, we express and purify from Escherichia coli the full-length LbpB and the N-lobe of LbpB from the bovine pathogen Moraxella bovis for crystallization trials. We demonstrate that M. bovis LbpB binds to bovine but not human Lf. We also report the crystal structure of the N-terminal lobe of LbpB from M. bovis and compare it with the published structures of TbpB to speculate on the process of Lf mediated iron acquisition.

scholarly journals Identification of Discrete Domains within Gonococcal Transferrin-Binding Protein A That Are Necessary for Ligand Binding and Iron Uptake Functions

2000 ◽  
Vol 68 (12) ◽  
pp. 6988-6996 ◽  
Author(s):  
Ian C. Boulton ◽  
Mary Kate Yost ◽  
James E. Anderson ◽  
Cynthia Nau Cornelissen
Keyword(s):  
Ligand Binding ◽  
Iron Uptake ◽  
Binding Protein ◽  
Protein A ◽  
Ligand Interaction ◽  
Whole Cells ◽  
Affinity Ligand ◽  
Discrete Domains ◽  
Transferrin Binding Proteins

ABSTRACT The availability of free iron in vivo is strictly limited, in part by the iron-binding protein transferrin. The pathogenicNeisseria spp. can sequester iron from this protein, dependent upon two iron-repressible, transferrin-binding proteins (TbpA and TbpB). TbpA is a TonB-dependent, integral, outer membrane protein that may form a β-barrel exposing multiple surface loops, some of which are likely to contain ligand-binding motifs. In this study we propose a topological model of gonococcal TbpA and then test some of the hypotheses set forth by the model by individually deleting three putative loops (designated loops 4, 5, and 8). Each mutant TbpA could be expressed without toxicity and was surface exposed as assessed by immunoblotting, transferrin binding, and protease accessibility. Deletion of loop 4 or loop 5 abolished transferrin binding to whole cells in solid- and liquid-phase assays, while deletion of loop 8 decreased the affinity of the receptor for transferrin without affecting the copy number. Strains expressing any of the three mutated TbpAs were incapable of growth on transferrin as a sole iron source. These data implicate putative loops 4 and 5 as critical determinants for receptor function and transferrin-iron uptake by gonococcal TbpA. The phenotype of the ΔL8TbpA mutant suggests that high-affinity ligand interaction is required for transferrin-iron internalization.

scholarly journals Expression and purification of functional recombinant meningococcal transferrin-binding protein A

2002 ◽  
Vol 364 (3) ◽  
pp. 613-616 ◽  
Author(s):  
Jonathan S. OAKHILL ◽  
Christopher L. JOANNOU ◽  
Susan K. BUCHANAN ◽  
Andrew R. GORRINGE ◽  
Robert W. EVANS
Keyword(s):  
Iron Uptake ◽  
Pathogenic Bacteria ◽  
Protein A ◽  
Direct Interaction ◽  
Uptake System ◽  
Meningococcal Vaccine ◽  
Expression And Purification ◽  
Receptor Interactions ◽  
Gated Channel ◽  
Transferrin Binding Proteins

Pathogenic bacteria of the genus Neisseria have a siderophore-independent iron-uptake system reliant on a direct interaction between the bacterial cell and human transferrin (hTf), a serum protein. In the meningococcus, this uptake system is dependent on two surface-exposed, transferrin-binding proteins (Tbps), TbpA and TbpB. TbpA is highly conserved among meningococcal strains, and is thought to be a porin-like integral protein that functions as a gated channel for the passage of iron into the periplasm. TbpB is more variable in size, lipidated and fully surface-exposed. Given its location on the cell surface, its role in pathogenicity and interstrain sequence conservation, TbpA is currently being regarded for inclusion in a meningococcal vaccine effective against all serogroups. This requires gaining knowledge of the ligand—receptor interactions. In the present study we have optimized a procedure for obtaining purified, functionally active recombinant TbpA at a level and stability necessary for the initiation of such studies.

scholarly journals Transferrin binding protein B and Transferrin binding protein A 2 expand the transferrin recognition range of Histophilus somni

2019 ◽  
Author(s):  
Anastassia K. Pogoutse ◽  
Trevor F. Moraes
Keyword(s):  
Host Species ◽  
Binding Protein ◽  
Iron Acquisition ◽  
Protein A ◽  
Acquisition System ◽  
Sheep And Goats ◽  
Iron Acquisition System ◽  
Histophilus Somni ◽  
Bovine Transferrin ◽  
Protein B

AbstractThe bacterial bipartite transferrin receptor is an iron acquisition system that is required for survival by several key human and animal pathogens. It consists of the TonB-dependent transporter Transferrin binding protein A (TbpA) and the surface lipoprotein Transferrin binding protein B (TbpB). Curiously, the Tbps are only found in host specific pathogens, and are themselves host specific, meaning that they will bind to the transferrin of their host species, but not to those of other animal species. While this phenomenon has long been established, neither the steps in the evolutionary process that led to this exquisite adaptation for the host, nor the steps that could alter it, are known. We sought to gain insight into these processes by studying Tbp specificity in Histophilus somni, a major pathogen of cattle. A past study showed that whole cells of H. somni specifically bind bovine transferrin, but not transferrin from sheep and goats, two bovids whose transferrins share 93% amino acid sequence identity with bovine transferrin. To our surprise, we found that H. somni can use sheep and goat transferrins as iron sources for growth, and that HsTbpB, but not HsTbpA, has detectable affinity for sheep and goat transferrins. Furthermore, a third transferrin binding protein, HsTbpA2, also showed affinity for sheep and goat transferrins. Our results show that H. somni TbpB and TbpA2 act to broaden the host transferrin recognition range of H. somni.ImportanceHost restricted pathogens infect a single host species or a narrow range of host species. Histophilus somni, a pathogen that incurs severe economic losses for the cattle industry, infects cattle, sheep, and goats, but not other mammals. The transferrin binding proteins, TbpA and TbpB, are thought to be a key iron acquisition system in H. somni, however, surprisingly, they were also shown to be cattle transferrin-specific. In our study we find that H. somni TbpB, and another little-studied Tbp, TbpA2, bind sheep and goat transferrins as well as bovine transferrin. Our results suggest that TbpA2 may have allowed for host range expansion, and provide a mechanism for how host specificity in Tbp containing pathogens can be altered.

The role of lactoferrin binding protein B in mediating protection against human lactoferricin1This article is part of a Special Issue entitled Lactoferrin and has undergone the Journal's usual peer review process.

2012 ◽  
Vol 90 (3) ◽  
pp. 417-423 ◽  
Author(s):  
Ari Morgenthau ◽  
Margaret Livingstone ◽  
Paul Adamiak ◽  
Anthony B. Schryvers
Keyword(s):  
Peer Review Process ◽  
Binding Protein ◽  
Iron Acquisition ◽  
Protein A ◽  
Human Lactoferrin ◽  
Cationic Antimicrobial Peptide ◽  
Iron Source ◽  
Iron Deficient ◽  
Protein B

Bacteria that inhabit the mucosal surfaces of the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment because of iron sequestration by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic, antimicrobial peptide lactoferricin is produced by proteolysis of lactoferrin. Several Gram-negative pathogens express a lactoferrin receptor that enables the bacteria to use lactoferrin as an iron source. The receptor is composed of an integral membrane protein, lactoferrin binding protein A (LbpA), and a membrane-bound lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for growth with lactoferrin as the sole iron source, whereas the role of LbpB in iron acquisition is not yet known. In this study, we demonstrate that LbpB from 2 different species is capable of providing protection against the killing activity of a human lactoferrin-derived peptide. We investigated the prevalence of lactoferrin receptors in bacteria and examined their sequence diversity. We propose that the protection against the cationic antimicrobial human lactoferrin-derived peptide is associated with clusters of negatively charged amino acids in the C-terminal lobe of LbpB that is a common feature of this protein.

scholarly journals Structural Basis for Iron Binding and Release by a Novel Class of Periplasmic Iron-Binding Proteins Found in Gram-Negative Pathogens

2004 ◽  
Vol 186 (12) ◽  
pp. 3903-3910 ◽  
Author(s):  
Stephen R. Shouldice ◽  
Robert J. Skene ◽  
Douglas R. Dougan ◽  
Gyorgy Snell ◽  
Duncan E. McRee ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Binding Protein ◽  
Iron Acquisition ◽  
Protein A ◽  
Structural Basis ◽  
Mammalian Host ◽  
Iron Binding ◽  
Gram Negative ◽  
Shipping Fever ◽  
Iron Binding Proteins

ABSTRACT We have determined the 1.35- and 1.45-Å structures, respectively, of closed and open iron-loaded forms of Mannheimia haemolytica ferric ion-binding protein A. M. haemolytica is the causative agent in the economically important and fatal disease of cattle termed shipping fever. The periplasmic iron-binding protein of this gram-negative bacterium, which has homologous counterparts in many other pathogenic species, performs a key role in iron acquisition from mammalian host serum iron transport proteins and is essential for the survival of the pathogen within the host. The ferric (Fe3+) ion in the closed structure is bound by a novel asymmetric constellation of four ligands, including a synergistic carbonate anion. The open structure is ligated by three tyrosyl residues and a dynamically disordered solvent-exposed anion. Our results clearly implicate the synergistic anion as the primary mediator of global protein conformation and provide detailed insights into the molecular mechanisms of iron binding and release in the periplasm.

scholarly journals The structure of lactoferrin-binding protein B fromNeisseria meningitidissuggests roles in iron acquisition and neutralization of host defences

2014 ◽  
Vol 70 (10) ◽  
pp. 1312-1317 ◽  
Author(s):  
Cory L. Brooks ◽  
Elena Arutyunova ◽  
M. Joanne Lemieux
Keyword(s):  
Binding Protein ◽  
Iron Acquisition ◽  
Protein A ◽  
Transport Systems ◽  
Cationic Peptide ◽  
Moraxella Bovis ◽  
Component Systems ◽  
Two Component Systems ◽  
Membrane Bound ◽  
Protein B

Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the generaNeisseriaandMoraxellahave evolved two-component systems that can extract iron from the host glycoproteins lactoferrin and transferrin. The homologous iron-transport systems consist of a membrane-bound transporter and an accessory lipoprotein. While the mechanism behind iron acquisition from transferrin is well understood, relatively little is known regarding how iron is extracted from lactoferrin. Here, the crystal structure of the N-terminal domain (N-lobe) of the accessory lipoprotein lactoferrin-binding protein B (LbpB) from the pathogenNeisseria meningitidisis reported. The structure is highly homologous to the previously determined structures of the accessory lipoprotein transferrin-binding protein B (TbpB) and LbpB from the bovine pathogenMoraxella bovis. Docking the LbpB structure with lactoferrin reveals extensive binding interactions with the N1 subdomain of lactoferrin. The nature of the interaction precludes apolactoferrin from binding LbpB, ensuring the specificity of iron-loaded lactoferrin. The specificity of LbpB safeguards proper delivery of iron-bound lactoferrin to the transporter lactoferrin-binding protein A (LbpA). The structure also reveals a possible secondary role for LbpB in protecting the bacteria from host defences. Following proteolytic digestion of lactoferrin, a cationic peptide derived from the N-terminus is released. This peptide, called lactoferricin, exhibits potent antimicrobial effects. The docked model of LbpB with lactoferrin reveals that LbpB interacts extensively with the N-terminal lactoferricin region. This may provide a venue for preventing the production of the peptide by proteolysis, or directly sequestering the peptide, protecting the bacteria from the toxic effects of lactoferricin.

Web-Based Bioinformatics Analysis of TbpB from Actinobacillus pleuropneumonie L20 Strain

2012 ◽  
Vol 466-467 ◽  
pp. 256-261
Author(s):  
Ren Feng Li ◽  
Xiang Qin Tian ◽  
Kun Zhao ◽  
Jin Qing Jiang ◽  
San Hu Wang
Keyword(s):  
Membrane Protein ◽  
Bioinformatics Analysis ◽  
Binding Protein ◽  
Iron Acquisition ◽  
Protein A ◽  
Actinobacillus Pleuropneumoniae ◽  
Biological Characterization ◽  
Web Based

The transferring(Tf) receptor from Actinobacillus pleuropneumoniae (App) is comprised of a surface exposed lipoprotein, Tf-binding protein B (TbpB), and an integral outer-membrane protein, Tf-binding protein A (TbpA), both of which are essential for survival in the host, and TbpB is required for the iron acquisition process in vivo. In this study. We analyzed the salient features of the TbpB gene and encoded protein of App L20 strain by bioinformatics tools and highlighted its important biological characterization, which. providing insights into the mechanism of Tf binding and the role of TbpB,

scholarly journals Makorin 1 controls embryonic patterning by alleviating Bruno1-mediated repression ofoskartranslation

2018 ◽  
Author(s):  
Annabelle Dold ◽  
Hong Han ◽  
Niankun Liu ◽  
Andrea Hildebrandt ◽  
Mirko Brüggemann ◽  
...  
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Binding Protein ◽  
Protein A ◽  
Cell Formation ◽  
Posterior Pole ◽  
Embryonic Patterning ◽  
Translational Repressor ◽  
Translational Activation ◽  
Translational Activator

AbstractMakorins are evolutionary conserved proteins that contain C3H-type zinc finger modules and a RING E3 ubiquitin ligase domain. InDrosophilamaternal Makorin 1 (Mkrn1) has been linked to embryonic patterning but the mechanism remained unsolved. Here, we show that Mkrn1 is essential for axis specification and pole plasm assembly by translational activation ofoskar. We demonstrate that Mkrn1 interacts with poly(A) binding protein (pAbp) and bindsosk3’ UTR in a region adjacent to A-rich sequences. This binding site overlaps with Bruno1 (Bru1) responsive elements (BREs), which regulateosktranslation. We observe increased association of the translational repressor Bru1 withoskmRNA upon depletion of Mkrn1, indicating that both proteins compete foroskbinding. Consistently, reducing Bru1 dosage partially rescues viability and Osk protein level in ovaries fromMkrn1females. We conclude that Mkrn1 controls embryonic patterning and germ cell formation by specifically activatingosktranslation by displacing Bru1 from its 3’ UTR.Author SummaryTo ensure accurate development of theDrosophilaembryo, proteins and mRNAs are positioned at specific sites within the embryo. Many of these proteins and mRNAs are produced and localized during the development of the egg in the mother. One protein essential for this process that has been heavily studied is Oskar (Osk), which is positioned at the posterior pole. During the localization ofoskmRNA, its translation is repressed by the RNA-binding protein Bruno1 (Bru1), ensuring that Osk protein is not present outside of the posterior where it is harmful. At the posterior pole,oskmRNA is activated through mechanisms that are not yet understood. In this work, we show that the conserved protein Makorin 1 (Mkrn1) is a novel factor involved in the translational activation ofosk. Mkrn1 binds specifically tooskmRNA in a region that overlaps with the binding site of Bru1, thus alleviating the association of Bru1 withosk. Moreover, Mkrn1 is stabilized by poly(A) binding protein, a translational activator that bindsoskmRNA in close proximity to Mkrn1. Our work thus helps to answer a long-standing question in the field, providing insight about the function of Mkrn1 and more generally into embryonic patterning in animals.

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