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.

• Keywords
• RTX toxins, cell adhesion, clip-and-link, host-pathogen interactions, nuclear magnetic resonance,
• MeSH
• Actinobacillus pleuropneumoniae chemistry   pathogenicity   MeSH
• Bacterial Proteins chemistry   genetics   MeSH
• Bacterial Toxins chemistry   MeSH
• Actinobacillus Infections veterinary   MeSH
• Membrane Proteins chemistry   MeSH
• Neisseria meningitidis chemistry   MeSH
• Protein Processing, Post-Translational * MeSH
• Swine MeSH
• Calcium metabolism   MeSH
• Virulence MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ApxIVA protein, Actinobacillus pleuropneumoniae MeSH Browser
• Bacterial Proteins MeSH
• Bacterial Toxins MeSH
• frpC protein, Neisseria meningitidis MeSH Browser
• Membrane Proteins MeSH
• Calcium 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.

• Keywords
• Calcium-binding site, Energy transfer, Excitation Spectrum, Protein fluorescence, Terbium phosphorescence, Tryptophan,
• Publication type
• Journal Article MeSH

The formation of mineralized tissues is governed by extracellular matrix proteins that assemble into a 3D organic matrix directing the deposition of hydroxyapatite. Although the formation of bones and dentin depends on the self-assembly of type I collagen via the Gly-X-Y motif, the molecular mechanism by which enamel matrix proteins (EMPs) assemble into the organic matrix remains poorly understood. Here we identified a Y/F-x-x-Y/L/F-x-Y/F motif, evolutionarily conserved from the first tetrapods to man, that is crucial for higher order structure self-assembly of the key intrinsically disordered EMPs, ameloblastin and amelogenin. Using targeted mutations in mice and high-resolution imaging, we show that impairment of ameloblastin self-assembly causes disorganization of the enamel organic matrix and yields enamel with disordered hydroxyapatite crystallites. These findings define a paradigm for the molecular mechanism by which the EMPs self-assemble into supramolecular structures and demonstrate that this process is crucial for organization of the organic matrix and formation of properly structured enamel.

• Keywords
• ameloblastin, amelogenin, biomineralization, enamel, intrinsically disordered protein,
• MeSH
• Amelogenin metabolism   MeSH
• Amino Acid Motifs physiology   MeSH
• Biological Evolution MeSH
• Extracellular Matrix Proteins metabolism   MeSH
• Durapatite metabolism   MeSH
• Mice MeSH
• Dental Enamel Proteins metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Binding physiology   MeSH
• Intrinsically Disordered Proteins metabolism   MeSH
• Dental Enamel metabolism   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Amelogenin MeSH
• enamel matrix proteins MeSH Browser
• Extracellular Matrix Proteins MeSH
• Durapatite MeSH
• Dental Enamel Proteins MeSH
• Intrinsically Disordered Proteins 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
• Bacterial Proteins chemistry   genetics   MeSH
• Cell Adhesion genetics   MeSH
• X-Ray Diffraction MeSH
• Humans MeSH
• Lipoproteins chemistry   metabolism   MeSH
• Membrane Proteins chemistry   genetics   MeSH
• Neisseria meningitidis chemistry   genetics   MeSH
• Periplasmic Binding Proteins chemistry   metabolism   MeSH
• Iron-Binding Proteins chemistry   metabolism   MeSH
• Bacterial Outer Membrane Proteins metabolism   MeSH
• Amino Acid Sequence genetics   MeSH
• Iron chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• frpC protein, Neisseria meningitidis MeSH Browser
• Lipoproteins MeSH
• Membrane Proteins MeSH
• Periplasmic Binding Proteins MeSH
• Iron-Binding Proteins MeSH
• Bacterial Outer Membrane Proteins MeSH
• Iron MeSH

This review summarizes the main results obtained in the fields of general and molecular microbiology and microbial genetics at the Institute of Microbiology of the Academy of Sciences of the Czech Republic (AS CR) [formerly Czechoslovak Academy of Sciences (CAS)] over more than 50 years. Contribution of the founder of the Institute, academician Ivan Málek, to the introduction of these topics into the scientific program of the Institute of Microbiology and to further development of these studies is also included.

• MeSH
• Academies and Institutes history   MeSH
• History, 20th Century MeSH
• Genetics, Microbial history   MeSH
• Molecular Biology history   MeSH
• Check Tag
• History, 20th Century MeSH
• Publication type
• Journal Article MeSH
• Historical Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Geographicals
• Czech Republic MeSH

Repeats-in-toxin (RTX) exoproteins of Gram-negative bacteria form a steadily growing family of proteins with diverse biological functions. Their common feature is the unique mode of export across the bacterial envelope via the type I secretion system and the characteristic, typically nonapeptide, glycine- and aspartate-rich repeats binding Ca(2+) ions. In this review, we summarize the current state of knowledge on the organization of rtx loci and on the biological and biochemical activities of therein encoded proteins. Applying several types of bioinformatic screens on the steadily growing set of sequenced bacterial genomes, over 1000 RTX family members were detected, with the biological functions of most of them remaining to be characterized. Activities of the so far characterized RTX family members are then discussed and classified according to functional categories, ranging from the historically first characterized pore-forming RTX leukotoxins, through the large multifunctional enzymatic toxins, bacteriocins, nodulation proteins, surface layer proteins, up to secreted hydrolytic enzymes exhibiting metalloprotease or lipase activities of industrial interest.

• MeSH
• Amino Acid Motifs MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Bacterial Toxins chemistry   genetics   metabolism   MeSH
• Gram-Negative Bacteria chemistry   genetics   metabolism   MeSH
• Multigene Family * MeSH
• Gene Expression Regulation, Bacterial MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Bacterial Toxins MeSH

Fe-regulated protein D (FrpD) is a Neisseria meningitidis outer membrane lipoprotein that may be involved in the anchoring of the secreted repeat in toxins (RTX) protein FrpC to the outer bacterial membrane. However, the function and biological roles of the FrpD and FrpC proteins remain unknown. Native and selenomethionine-substituted variants of recombinant FrpD43-271 protein were crystallized using the sitting-drop vapour-diffusion method. Diffraction data were collected to a resolution of 2.25 A for native FrpD43-271 protein and to a resolution of 2.00 A for selenomethionine-substituted FrpD43-271 (SeMet FrpD43-271) protein. The crystals of native FrpD43-271 protein belonged to the hexagonal space group P6(2) or P6(4), while the crystals of SeMet FrpD43-271 protein belonged to the primitive orthorhombic space group P2(1)2(1)2(1).

• MeSH
• Crystallization MeSH
• Crystallography, X-Ray MeSH
• Lipoproteins chemistry   MeSH
• Neisseria meningitidis chemistry   MeSH
• Bacterial Outer Membrane Proteins chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lipoproteins MeSH
• Bacterial Outer Membrane Proteins MeSH

Purification of recombinant proteins is often a challenging process involving several chromatographic steps that must be optimized for each target protein. Here, we developed a self-excising module allowing single-step affinity chromatography purification of untagged recombinant proteins. It consists of a 250-residue-long self-processing module of the Neisseria meningitidis FrpC protein with a C-terminal affinity tag. The N terminus of the module is fused to the C terminus of a target protein of interest. Upon binding of the fusion protein to an affinity matrix from cell lysate and washing out contaminating proteins, site-specific cleavage of the Asp-Pro bond linking the target protein to the self-excising module is induced by calcium ions. This results in the release of the target protein with only a single aspartic acid residue added at the C terminus, while the self-excising affinity module remains trapped on the affinity matrix. The system was successfully tested with several target proteins, including glutathione-S-transferase, maltose-binding protein, beta-galactosidase, chloramphenicol acetyltransferase, and adenylate cyclase, and two different affinity tags, chitin-binding domain or poly-His. Moreover, it was demonstrated that it can be applied as an alternative to two currently existing systems, based on the self-splicing intein of Saccharomyces cerevisiae and sortase A of Staphylococcus aureus.

• MeSH
• Bacterial Proteins chemistry   MeSH
• Chromatography, Affinity methods   MeSH
• Aspartic Acid chemistry   MeSH
• Membrane Proteins chemistry   MeSH
• Recombinant Proteins chemistry   isolation & purification   MeSH
• Protein Structure, Tertiary MeSH
• Calcium chemistry   MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• frpC protein, Neisseria meningitidis MeSH Browser
• Aspartic Acid MeSH
• Membrane Proteins MeSH
• Recombinant Proteins MeSH
• Calcium MeSH