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

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.

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Crystal Structure ofPasteurella haemolyticaFerric Ion-binding Protein A Reveals a Novel Class of Bacterial Iron-binding Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.m306821200 ◽

2003 ◽

Vol 278 (42) ◽

pp. 41093-41098 ◽

Cited By ~ 22

Author(s):

Stephen R. Shouldice ◽

Douglas R. Dougan ◽

Pamela A. Williams ◽

Robert J. Skene ◽

Gyorgy Snell ◽

...

Keyword(s):

Crystal Structure ◽

Binding Proteins ◽

Binding Protein ◽

Protein A ◽

Ion Binding ◽

Iron Binding ◽

Iron Binding Proteins

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Bacterial lactoferrin-binding protein A binds to both domains of the human lactoferrin C-lobe

Microbiology ◽

10.1099/mic.0.26281-0 ◽

2003 ◽

Vol 149 (7) ◽

pp. 1729-1737 ◽

Cited By ~ 15

Author(s):

Henry Wong ◽

Anthony B. Schryvers

Keyword(s):

Binding Proteins ◽

Binding Protein ◽

Protein A ◽

Human Lactoferrin ◽

Chimeric Proteins ◽

Iron Binding ◽

Binding Studies ◽

Binding Interaction ◽

Binding Regions ◽

Iron Binding Proteins

Pathogenic bacteria in the family Neisseriaceae express surface receptors to acquire iron from the mammalian iron-binding proteins. Transferrins and lactoferrins constitute a family of iron-binding proteins highly related in both sequence and structure, yet the bacterial receptors are able to distinguish between these proteins and uphold a strict binding specificity. In order to understand the molecular basis for this specificity, the interaction between human lactoferrin (hLf) and the lactoferrin-binding protein A (LbpA) from Moraxella catarrhalis was studied. A periplasmic expression system was designed for the heterologous expression of LbpA, which enabled the investigation of its binding activity in the absence of lactoferrin-binding protein B (LbpB). To facilitate delineation of the LbpA-binding regions of hLf, chimeric proteins composed of hLf and bovine transferrin were made. Binding studies performed with the chimeric proteins and recombinant LbpA identified two binding regions within the C-terminus of hLf. Furthermore, native LbpA from Moraxella and Neisseria spp. bound the identical spectrum of hybrid proteins as the recombinant receptor, demonstrating a conserved binding interaction with the C-lobe of hLf.

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An Immunoreactive 38-Kilodalton Protein of Ehrlichia canis Shares Structural Homology and Iron-Binding Capacity with the Ferric Ion-Binding Protein Family

Infection and Immunity ◽

10.1128/iai.73.1.62-69.2005 ◽

2005 ◽

Vol 73 (1) ◽

pp. 62-69 ◽

Cited By ~ 17

Author(s):

C. Kuyler Doyle ◽

Xiaofeng Zhang ◽

Vsevolod L. Popov ◽

Jere W. McBride

Keyword(s):

Binding Capacity ◽

Binding Protein ◽

Iron Acquisition ◽

Ehrlichia Canis ◽

Dimensional Structure ◽

Ion Binding ◽

Ferric Ion ◽

Gram Negative Bacteria ◽

Iron Binding ◽

Gram Negative

ABSTRACT Ehrlichiae are tick-transmitted, gram-negative, obligately intracellular bacteria that live and replicate in cytoplasmic vacuoles, but little is known about iron acquisition mechanisms necessary for their survival. In this study, a genus-conserved immunoreactive ferric ion-binding protein (Fbp) of Ehrlichia canis was identified and its iron-binding capability was investigated. E. canis Fbp was homologous to a family of periplasmic Fbp's involved in iron acquisition and transport in gram-negative bacteria. E. canis Fbp had a molecular mass (38 kDa) consistent with those of Fbp's in other bacteria and exhibited substantial immunoreactivity in its native conformation. The predicted three-dimensional structure of E. canis Fbp demonstrated conservation of important Fbp family structural motifs: two domains linked with a polypeptide “hinge” region. Under iron-binding conditions, the recombinant Fbp exhibited an intense red color and an absorbance spectrum indicative of iron binding, and it bound Fe(III) but not Fe(II). Fbp was observed primarily in the cytoplasm of the reticulate forms of E. canis and Ehrlichia chaffeensis but was notably found on extracellular morula fibers in morulae containing dense-cored organisms. Although expression of Fbp is regulated through an operon of three functionally linked genes in other gram-negative bacteria, the absence of an intact fbp operon in Ehrlichia spp. suggests that genes involved in ehrlichial iron acquisition have been subject to reductive evolution.

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Human C4b-binding Protein, Structural Basis for Interaction with Streptococcal M Protein, a Major Bacterial Virulence Factor

Journal of Biological Chemistry ◽

10.1074/jbc.m511563200 ◽

2005 ◽

Vol 281 (6) ◽

pp. 3690-3697 ◽

Cited By ~ 41

Author(s):

Huw T. Jenkins ◽

Linda Mark ◽

Graeme Ball ◽

Jenny Persson ◽

Gunnar Lindahl ◽

...

Keyword(s):

Virulence Factor ◽

Binding Protein ◽

Protein A ◽

Bacterial Virulence ◽

M Protein ◽

Structural Basis ◽

Streptococcal M Protein ◽

Bacterial Virulence Factor

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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.

Biochemistry and Cell Biology ◽

10.1139/o11-078 ◽

2012 ◽

Vol 90 (3) ◽

pp. 351-361 ◽

Cited By ~ 7

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.

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Transcription Factor 21 Promotes Chicken Adipocyte Differentiation at Least in Part via Activating MAPK/JNK Signaling

Genes ◽

10.3390/genes12121971 ◽

2021 ◽

Vol 12 (12) ◽

pp. 1971

Author(s):

Xinyang Zhang ◽

Bohan Cheng ◽

Haixu Jiang ◽

Chang Liu ◽

Zhiping Cao ◽

...

Keyword(s):

Transcription Factor ◽

Cell Line ◽

Molecular Mechanisms ◽

Binding Protein ◽

Protein A ◽

Peroxisome Proliferator ◽

Jnk Pathway ◽

Peroxisome Proliferator Activated Receptor ◽

Fatty Acid Binding ◽

Enhancer Binding Protein

The molecular mechanisms of transcription factor 21 (TCF21) in regulating chicken adipogenesis remain unclear. Thus, the current study was designed to investigate the signaling pathway mediating the effect of TCF21 on chicken adipogenesis. Immortalized chicken preadipocytes cell line (ICP), a preadipocyte cell line stably overexpressing TCF21 (LV-TCF21) and a control preadipocyte cell line (LV-control) were used in the current study. We found that the phosphorylation of c-Jun N-terminal kinases (JNK) was significantly elevated in LV-TCF21 compared to LV-control. After treating ICP cells with a JNK inhibitor SP600125, the differentiation of ICP was inhibited, as evidenced by decreased accumulation of lipid droplets and reduced expression of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein α (C/EBPα), adipocyte fatty acid binding protein (A-FABP), and lipoprotein lipase (LPL). Moreover, we found that the inhibition of JNK by SP600125 remarkably impaired the ability of TCF21 to drive adipogenesis. Taken together, our results suggest that TCF21 promotes the differentiation of adipocytes at least in part via activating MAPK/JNK pathway.

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

10.1101/730739 ◽

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.

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Preparation and Characterization of Neisseria meningitidis Mutants Deficient in Production of the Human Lactoferrin-Binding Proteins LbpA and LbpB

Journal of Bacteriology ◽

10.1128/jb.180.12.3080-3090.1998 ◽

1998 ◽

Vol 180 (12) ◽

pp. 3080-3090 ◽

Cited By ~ 35

Author(s):

Robert A. Bonnah ◽

Anthony B. Schryvers

Keyword(s):

Neisseria Meningitidis ◽

Binding Proteins ◽

Binding Protein ◽

Iron Acquisition ◽

Outer Membrane Proteins ◽

Mammalian Host ◽

Human Pathogens ◽

Sequence Comparisons ◽

Isolation Technique

ABSTRACT Pathogenic members of the family Neisseriaceae produce specific receptors facilitating iron acquisition from transferrin (Tf) and lactoferrin (Lf) of their mammalian host. Tf receptors are composed of two outer membrane proteins, Tf-binding proteins A and B (TbpA and TbpB; formerly designated Tbp1 and Tbp2, respectively). Although only a single Lf-binding protein, LbpA (formerly designated Lbp1), had previously been recognized, we recently identified additional bacterial Lf-binding proteins in the human pathogens Neisseria meningitidis and Moraxella catarrhalis and the bovine pathogen Moraxella bovis by a modified affinity isolation technique (R. A. Bonnah, R.-H. Yu, and A. B. Schryvers, Microb. Pathog. 19:285–297, 1995). In this report, we characterize an open reading frame (ORF) located immediately upstream of theN. meningitidis B16B6 lbpA gene. Amino acid sequence comparisons of various TbpBs with the product of the translated DNA sequence from the upstream ORF suggests that the region encodes the Lf-binding protein B homolog (LbpB). The LbpB from strain B16B6 has two large stretches of negatively charged amino acids that are not present in the various transferrin receptor homologs (TbpBs). Expression of the recombinant LbpB protein as a fusion with maltose binding protein demonstrated functional Lf-binding activity. Studies with N. meningitidis isogenic mutants in which thelbpA gene and the ORF immediately upstream oflbpA (putative lbpB gene) were insertionally inactivated demonstrated that LbpA, but not LbpB, is essential for iron acquisition from Lf in vitro.

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Plasmodium sporozoites require the protein B9 to invade hepatocytes

10.1101/2021.10.25.465731 ◽

2021 ◽

Author(s):

Priyanka Fernandes ◽

Manon Loubens ◽

Carine Marinach ◽

Romain Coppee ◽

Morgane Grand ◽

...

Keyword(s):

Protein Function ◽

Molecular Mechanisms ◽

Protein Complexes ◽

Protein A ◽

Blood Feeding ◽

Host Cells ◽

Infected Mosquito ◽

Mammalian Host ◽

Genetic Tagging ◽

Structural Levels

Plasmodium sporozoites are transmitted to a mammalian host during blood feeding by an infected mosquito and invade hepatocytes for initial replication of the parasite in the liver. This leads to the release of thousands of merozoites into the blood circulation and initiation of the pathogenic blood stages of malaria. Merozoite invasion of erythrocytes has been well characterized at the molecular and structural levels. In sharp contrast, the molecular mechanisms of sporozoite invasion of hepatocytes are poorly characterized. Here we report a new role during sporozoite entry for the B9 protein, a member of the 6-cysteine domain protein family. Using genetic tagging and gene deletion approaches in rodent malaria parasites, we show that B9 is secreted from sporozoite micronemes and is required for productive invasion of hepatocytes. Structural modelling indicates that the N-terminus of B9 forms a beta-propeller domain structurally related to CyRPA, a cysteine-rich protein forming an invasion complex with Rh5 and RIPR in P. falciparum merozoites. We provide evidence that the beta-propeller domain of B9 is essential for protein function during sporozoite entry and interacts with P36 and P52, both also essential for productive invasion of hepatocytes. Our results suggest that, despite using distinct sets of parasite and host entry factors, Plasmodium sporozoites and merozoites may share common structural modules to assemble protein complexes for invasion of host cells.

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