Structural Variations within the Transferrin Binding Site on Transferrin-binding Protein B, TbpB

Calmettes, Charles; Yu, Rong Hua; Silva, Leslie P.; Curran, Dave; Schriemer, David C.; Schryvers, Anthony B.; Moraes, Trevor F.

doi:10.1074/jbc.m110.206102

Cited by 44 publications

(56 citation statements)

References 31 publications

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“…Surface Plasmon Resonance-Surface Plasmon resonance experiments were performed using a BiacoreX instrument (GE Healthcare) at 25°C, similar to those described by Calmettes et al (31). Briefly, four pTf constructs (wild-type and pTf mutants D360A, R509A, and S625K) were coupled to the sensor chip (research-grade CM5) via standard N-hydroxysuccinimide and N-ethyl-N-(dimethylaminopropyl) carbodiimide activation.…”

Section: Methodsmentioning

confidence: 99%

“…Rosetta Docking-The Rosetta models used in this paper were generated as described by Moraes et al (5) and Calmettes et al (31). Briefly, the RosettaDock program was run in full atom mode using standard Monte Carlo movements, allowing for spin around the axis connecting the two proteins.…”

Section: Methodsmentioning

confidence: 99%

“…Decoys that failed on any important residue were deleted. The program was then run continuously until all decoys met each of the designated TbpB distance constraints (31).…”

Section: Methodsmentioning

confidence: 99%

“…Recent work by Ling et al has described two TbpB variants from Neisseria meningitidis that interact with the C lobe of human transferrin (hTf) (11), although it has been previously reported that both Tf lobes are involved in binding, specifically for Moraxella catarrhalis (23) and the ovine pathogen Haemophilus paragallinarum (30). By taking advantage of structural information available for divergent TbpBs from porcine pathogens (5,31), the current study was initiated to determine how the considerable variability in the TbpB surface regions maintain the interaction with pTf. Together with a companion study by Calmettes et al (31), we provide a structural analysis of the receptor-Tf interaction representing several divergent porcine pathogenic strains and evaluate the impact of iron loading on the molecular recognition mechanism in greater detail.…”

mentioning

confidence: 99%

“…In the current study, shift mapping was used to locate interfaces and altered dynamics upon the binding of pTf to four TbpB variants. These mapping data were used to test heterodimer models arising from computational docking of pTf to available TbpB structures (5,31). The models were further tested through mutations and surface plasmon resonance binding studies, as guided by hot spot analysis.…”

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confidence: 99%

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Conserved Interaction between Transferrin and Transferrin-binding Proteins from Porcine Pathogens

Silva

Calmettes

et al. 2011

Journal of Biological Chemistry

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Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions. Bacteria are dependent upon effective and efficient iron acquisition mechanisms to survive and proliferate in the ironlimited environment of the host (1-3). Pathogenic Gram-negative bacteria within the Neisseriaceae and Pasteurellaceae families rely on specialized uptake systems to acquire iron directly from host iron-binding proteins (2-5). These bacteria can acquire iron from host transferrin (Tf) 3 in particular (6), but in some cases also lactoferrin (7, 8) and hemogobulin/haptoglobulin (9). To understand such uptake mechanisms, a detailed characterization of relevant receptor-host protein interactions is required.The bacterial Tf receptors are composed of two iron-repressible surface components, transferrin binding protein A (TbpA), a TonB-dependent integral outer membrane protein of ϳ100 kDa, and transferrin-binding protein B (TbpB), a lipoprotein varying in size from 60 to 100 kDa (2, 3, 5, 10, 11). They have been found in clinical isolates of the Neisseriaceae, Pasteurellaceae, as well as the Moraxellaceae families (12) and constitute the outer membrane receptor complex responsible for binding Tf and transporting iron across the outer membrane into the periplasmic space (4, 13). These receptors exhibit a strict host specificity. For example, receptors of porcine pathogens will specifically bind porcine transferrin (pTf) but not human, avian, bovine, or ovine Tf (14).Tf is a glycoprotein of ϳ80 kDa, composed of two highly homologous N and C lobes (15). Each lobe contains two domains, connected by two antiparallel ␤-strands in a clamshell-like fold, producing a cleft that coordinates Fe 3ϩ in the binding pocket, along with a synergistic anion (CO 3 2Ϫ or C 2 O 4 ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Decoys that failed on any important residue were deleted. The program was then run continuously until all decoys met each of the designated TbpB distance constraints (31).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Conserved Interaction between Transferrin and Transferrin-binding Proteins from Porcine Pathogens

Silva

Calmettes

et al. 2011

Journal of Biological Chemistry

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Semiautomated design and soluble expression of a chimeric antigen TbpAB01 from Glaesserella parasuis

Chen,

Zhou,

Gong

et al. 2024

Biotech & Bioengineering

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Pathogenic bacterial membrane proteins (MPs) are a class of vaccine and antibiotic development targets with widespread clinical application. However, the inherent hydrophobicity of MPs poses a challenge to fold correctly in living cells. Herein, we present a comprehensive method to improve the soluble form of MP antigen by rationally designing multi‐epitope chimeric antigen (ChA) and screening two classes of protein‐assisting folding element. The study uses a homologous protein antigen as a functional scaffold to generate a ChA possessing four epitopes from transferrin‐binding protein A of Glaesserella parasuis. Our engineered strain, which co‐expresses P17 tagged‐ChA and endogenous chaperones groEL‐ES, yields a 0.346 g/L highly soluble ChA with the property of HPS‐positive serum reaction. Moreover, the protein titer of ChA reaches 4.27 g/L with >90% soluble proportion in 5‐L bioreactor, which is the highest titer reported so far. The results highlight a timely approach to design and improve the soluble expression of MP antigen in industrially viable applications.

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Patterns of structural and sequence variation within isotype lineages of the Neisseria meningitidis transferrin receptor system

et al. 2015

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Neisseria meningitidis inhabits the human upper respiratory tract and is an important cause of sepsis and meningitis. A surface receptor comprised of transferrin-binding proteins A and B (TbpA and TbpB), is responsible for acquiring iron from host transferrin. Sequence and immunological diversity divides TbpBs into two distinct lineages; isotype I and isotype II. Two representative isotype I and II strains, B16B6 and M982, differ in their dependence on TbpB for in vitro growth on exogenous transferrin. The crystal structure of TbpB and a structural model for TbpA from the representative isotype I N. meningitidis strain B16B6 were obtained. The structures were integrated with a comprehensive analysis of the sequence diversity of these proteins to probe for potential functional differences. A distinct isotype I TbpA was identified that co-varied with TbpB and lacked sequence in the region for the loop 3 α-helix that is proposed to be involved in iron removal from transferrin. The tightly associated isotype I TbpBs had a distinct anchor peptide region, a distinct, smaller linker region between the lobes and lacked the large loops in the isotype II C-lobe. Sequences of the intact TbpB, the TbpB N-lobe, the TbpB C-lobe, and TbpA were subjected to phylogenetic analyses. The phylogenetic clustering of TbpA and the TbpB C-lobe were similar with two main branches comprising the isotype 1 and isotype 2 TbpBs, possibly suggesting an association between TbpA and the TbpB C-lobe. The intact TbpB and TbpB N-lobe had 4 main branches, one consisting of the isotype 1 TbpBs. One isotype 2 TbpB cluster appeared to consist of isotype 1 N-lobe sequences and isotype 2 C-lobe sequences, indicating the swapping of N-lobes and C-lobes. Our findings should inform future studies on the interaction between TbpB and TbpA and the process of iron acquisition.

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Structural Variations within the Transferrin Binding Site on Transferrin-binding Protein B, TbpB

Cited by 44 publications

References 31 publications

Conserved Interaction between Transferrin and Transferrin-binding Proteins from Porcine Pathogens

Conserved Interaction between Transferrin and Transferrin-binding Proteins from Porcine Pathogens

Semiautomated design and soluble expression of a chimeric antigen TbpAB01 from Glaesserella parasuis

Patterns of structural and sequence variation within isotype lineages of the Neisseria meningitidis transferrin receptor system

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