Dataset Information

YopT domain of the PfhB2 toxin from Pasteurella multocida: protein expression, characterization, crystallization and crystallographic analysis.

ABSTRACT: Pasteurella multocida causes respiratory-tract infections in a broad range of animals, as well as opportunistic infections in humans. P. multocida secretes a multidomain toxin called PfhB2, which contains a YopT-like cysteine protease domain at its C-terminus. The YopT domain of PfhB2 contains a well conserved Cys-His-Asp catalytic triad that defines YopT family members, and shares high sequence similarity with the prototype YopT from Yersinia sp. To date, only one crystal structure of a YopT family member has been reported; however, additional structural information is needed to help characterize the varied substrate specificity and enzymatic action of this large protease family. Here, a catalytically inactive C3733S mutant of PfhB2 YopT that provides enhanced protein stability was used with the aim of gaining structural insight into the diversity within the YopT protein family. To this end, the C3733S mutant of PfhB2 YopT has been successfully cloned, overexpressed, purified and crystallized. Diffraction data sets were collected from native crystals to 3.5?Å resolution and a single-wavelength anomalous data set was collected from an iodide-derivative crystal to 3.2?Å resolution. Data pertaining to crystals belonging to space group P31, with unit-cell parameters a = 136.9, b = 136.9, c = 74.7?Å for the native crystals and a = 139.2, b = 139.2, c = 74.7?Å for the iodide-derivative crystals, are discussed.

SUBMITTER: Kumar S

PROVIDER: S-EPMC5947697 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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YopT domain of the PfhB2 toxin from Pasteurella multocida: protein expression, characterization, crystallization and crystallographic analysis.

Kumar Sanjeev S Hedrick Victoria V Mattoo Seema S

Acta crystallographica. Section F, Structural biology communications 20180226 Pt 3

Pasteurella multocida causes respiratory-tract infections in a broad range of animals, as well as opportunistic infections in humans. P. multocida secretes a multidomain toxin called PfhB2, which contains a YopT-like cysteine protease domain at its C-terminus. The YopT domain of PfhB2 contains a well conserved Cys-His-Asp catalytic triad that defines YopT family members, and shares high sequence similarity with the prototype YopT from Yersinia sp. To date, only one crystal structure of a YopT fa ...[more]

PMID: 29497015

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Project description:Bone remodeling is a continuous process to retain the structural integrity and function of the skeleton. A tight coupling is maintained between osteoclast-mediated resorption of old or damaged bones and osteoblast-mediated formation of new bones for bone homeostasis. While osteoblasts differentiate from mesenchymal stem cells, osteoclasts are hematopoietic in origin and derived from myeloid precursor cells. Osteoclast differentiation is driven by two cytokines, cytokine receptor activator of NF-κB ligand (RANKL), and macrophage colony-stimulating factor. Imbalances in the activity of osteoblasts and osteoclasts result in the development of bone disorders. Bacterially caused porcine atrophic rhinitis is characterized by a loss of nasal ventral conche bones and a distortion of the snout. While Bordetella bronchiseptica strains cause mild and reversible symptoms, infection of pigs with toxigenic Pasteurella multocida strains causes a severe and irreversible decay. The responsible virulence factor Pasteurella multocida toxin (PMT) contains a deamidase activity in its catalytical domain that constitutively activates specific heterotrimeric G proteins to induce downstream signaling cascades. While osteoblasts are inhibited by the toxin, osteoclasts are activated, thus skewing bone remodeling toward excessive bone degradation. Still, the mechanism by which PMT interferes with bone homeostasis, and the reason for this unusual target tissue is not yet well understood. Here, we show that PMT has the potential to differentiate bone marrow-derived macrophages into functional osteoclasts. This toxin-mediated differentiation process is independent of RANKL, a cytokine believed to be indispensable for triggering osteoclastogenesis, as addition of osteoprotegerin to PMT-treated macrophages does not show any effect on PMT-induced osteoclast formation. Although RANKL is not a prerequisite, toxin-primed macrophages show enhanced responsiveness to low concentrations of RANKL, suggesting that the PMT-generated microenvironment offers conditions where low concentrations of RANKL lead to an increase in the number of osteoclasts resulting in increased resorption. PMT-mediated release of the osteoclastogenic cytokines such as IL-6 and TNF-α, but not IL-1, supports the differentiation process. Although the production of cytokines and the subsequent activation of signaling cascades are necessary for PMT-mediated differentiation into osteoclasts, they are not sufficient and PMT-induced activation of G protein signaling is essential for efficient osteoclastogenesis.

Dataset Information

YopT domain of the PfhB2 toxin from Pasteurella multocida: protein expression, characterization, crystallization and crystallographic analysis.

Publications

YopT domain of the PfhB2 toxin from Pasteurella multocida: protein expression, characterization, crystallization and crystallographic analysis.

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