The posttranslational Ca2+-dependent "clip-and-link" activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a "twisted-amide" activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.IMPORTANCE The Ca2+-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2+-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca2+-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca2+-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.

• Klíčová slova
• RTX toxins, cell adhesion, clip-and-link, host-pathogen interactions, nuclear magnetic resonance,
• MeSH
• Actinobacillus pleuropneumoniae chemie   patogenita   MeSH
• bakteriální proteiny chemie   genetika   MeSH
• bakteriální toxiny chemie   MeSH
• infekce bakteriemi rodu Actinobacillus veterinární   MeSH
• membránové proteiny chemie   MeSH
• Neisseria meningitidis chemie   MeSH
• posttranslační úpravy proteinů * MeSH
• prasata MeSH
• vápník metabolismus   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ApxIVA protein, Actinobacillus pleuropneumoniae MeSH Prohlížeč
• bakteriální proteiny MeSH
• bakteriální toxiny MeSH
• frpC protein, Neisseria meningitidis MeSH Prohlížeč
• membránové proteiny MeSH
• vápník MeSH

The sensitized phosphorescence of Tb3+ is often used for the assessment of the ion binding to various chelating agents or natural Ca2+-binding proteins. The detailed structure of the Tb3+ excitation spectrum gives a special advantage for analysis; any extra absorption peak can be easily detected which provides simple and direct evidence that resonance energy transfer occurs. By employing the Tb3+ phosphorescence, we characterized the Ca2+-binding sites of two related peptides - self-processing module of the FrpC protein produced by bacterium Neisseria meningitidis and the shorter peptide derived from FrpC. Here we show that while the increase of direct Tb3+ excitation at 243 nm generally corresponds to Tb3+ association with various binding sites, the excitation enhancement in the 250-300 nm band signifies Tb3+-binding in the close proximity of aromatic residues. We demonstrate that the presence of resonance energy transfer could be easily detected by inspecting Tb3+ excitation spectra. Additionally, we show that the high level of specificity of Tb3+ steady state detection on the spectral level could be reached at very low Tb3+ concentrations by taking advantage of its narrow phosphorescence emission maximum at 545 nm and subtracting the averaged autofluorescence intensities outside this peak, namely at 525 and 565 nm.

• Klíčová slova
• Calcium-binding site, Energy transfer, Excitation Spectrum, Protein fluorescence, Terbium phosphorescence, Tryptophan,
• Publikační typ
• časopisecké články MeSH

The iron-regulated protein FrpD from Neisseria meningitidis is an outer membrane lipoprotein that interacts with very high affinity (Kd ~ 0.2 nM) with the N-terminal domain of FrpC, a Type I-secreted protein from the Repeat in ToXin (RTX) protein family. In the presence of Ca2+, FrpC undergoes Ca2+ -dependent protein trans-splicing that includes an autocatalytic cleavage of the Asp414-Pro415 peptide bond and formation of an Asp414-Lys isopeptide bond. Here, we report the high-resolution structure of FrpD and describe the structure-function relationships underlying the interaction between FrpD and FrpC1-414. We identified FrpD residues involved in FrpC1-414 binding, which enabled localization of FrpD within the low-resolution SAXS model of the FrpD-FrpC1-414 complex. Moreover, the trans-splicing activity of FrpC resulted in covalent linkage of the FrpC1-414 fragment to plasma membrane proteins of epithelial cells in vitro, suggesting that formation of the FrpD-FrpC1-414 complex may be involved in the interaction of meningococci with the host cell surface.

• MeSH
• bakteriální proteiny chemie   genetika   MeSH
• buněčná adheze genetika   MeSH
• difrakce rentgenového záření MeSH
• lidé MeSH
• lipoproteiny chemie   metabolismus   MeSH
• membránové proteiny chemie   genetika   MeSH
• Neisseria meningitidis chemie   genetika   MeSH
• periplazmatické vazebné proteiny chemie   metabolismus   MeSH
• proteiny vázající železo chemie   metabolismus   MeSH
• proteiny vnější bakteriální membrány metabolismus   MeSH
• sekvence aminokyselin genetika   MeSH
• železo chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• frpC protein, Neisseria meningitidis MeSH Prohlížeč
• lipoproteiny MeSH
• membránové proteiny MeSH
• periplazmatické vazebné proteiny MeSH
• proteiny vázající železo MeSH
• proteiny vnější bakteriální membrány MeSH
• železo MeSH