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 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

The transient receptor potential (TRP) protein superfamily consists of seven major groups, among them the "canonical TRP" family. The TRPC proteins are calcium-permeable nonselective cation channels activated after the emptying of intracellular calcium stores and appear to be gated by various types of messengers. The TRPC6 channel has been shown to be expressed in various tissues and cells, where it modulates the calcium level in response to external signals. Calcium binding proteins such as Calmodulin or the family of S100A proteins are regulators of TRPC channels. Here we characterized the overlapping integrative binding site for S100A1 at the C-tail of TRPC6, which is also able to accomodate various ligands such as Calmodulin and phosphatidyl-inositol-(4,5)-bisphosphate. Several positively charged amino acid residues (Arg852, Lys856, Lys859, Arg860 and Arg864) were determined by fluorescence anisotropy measurements for their participation in the calcium-dependent binding of S100A1 to the C terminus of TRPC6. The triple mutation Arg852/Lys859/Arg860 exhibited significant disruption of the binding of S100A1 to TRPC6. This indicates a unique involvement of these three basic residues in the integrative overlapping binding site for S100A1 on the C tail of TRPC6.

• MeSH
• anizotropie MeSH
• cirkulární dichroismus MeSH
• interakční proteinové domény a motivy MeSH
• kationtové kanály TRPC chemie   genetika   MeSH
• kationtový kanál TRPC6 MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• proteiny S100 chemie   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• substituce aminokyselin MeSH
• vápník chemie   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kationtové kanály TRPC MeSH
• kationtový kanál TRPC6 MeSH
• proteiny S100 MeSH
• S100A1 protein MeSH Prohlížeč
• TRPC6 protein, human MeSH Prohlížeč
• vápník MeSH

Phosducin (Pdc), a highly conserved phosphoprotein, plays an important role in the regulation of G protein signaling, transcriptional control, and modulation of blood pressure. Pdc is negatively regulated by phosphorylation followed by binding to the 14-3-3 protein, whose role is still unclear. To gain insight into the role of 14-3-3 in the regulation of Pdc function, we studied structural changes of Pdc induced by phosphorylation and 14-3-3 protein binding using time-resolved fluorescence spectroscopy. Our data show that the phosphorylation of the N-terminal domain of Pdc at Ser-54 and Ser-73 affects the structure of the whole Pdc molecule. Complex formation with 14-3-3 reduces the flexibility of both the N- and C-terminal domains of phosphorylated Pdc, as determined by time-resolved tryptophan and dansyl fluorescence. Therefore, our data suggest that phosphorylated Pdc undergoes a conformational change when binding to 14-3-3. These changes involve the G(t)βγ binding surface within the N-terminal domain of Pdc, and thus could explain the inhibitory effect of 14-3-3 on Pdc function.

• MeSH
• fluorescenční spektrometrie MeSH
• fosfatidylcholiny MeSH
• fosfoproteiny chemie   metabolismus   MeSH
• fosforylace MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• oční proteiny chemie   metabolismus   MeSH
• proteiny 14-3-3 metabolismus   MeSH
• proteiny vázající GTP - regulátory chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• serin metabolismus   MeSH
• terciární struktura proteinů MeSH
• tryptofan MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-myristoyl-2-(12-((5-dimethylamino-1-naphthalenesulfonyl)amino)dodecanoyl)-sn-glycero-3-phosphocholine MeSH Prohlížeč
• fosfatidylcholiny MeSH
• fosfoproteiny MeSH
• oční proteiny MeSH
• phosducin MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• proteiny vázající GTP - regulátory MeSH
• serin MeSH
• tryptofan MeSH
• YWHAZ protein, human MeSH Prohlížeč

Transient receptor potential melastatin 3 ion channel (TRPM3) belongs to the TRP family of cation-permeable ion channels involved in many important biological functions such as pain transduction, thermosensation, and mechanoregulation. The channel was reported to play an important role in Ca(2+) homeostasis, but its gating mechanisms, functions, and regulation are still under research. Utilizing biophysical and biochemical methods, we characterized two independent domains, Ala-35-Lys-124 and His-291-Gly-382, on the TRPM3 N terminus, responsible for interactions with the Ca(2+)-binding proteins calmodulin (CaM) and S100A1. We identified several positively charged residues within these domains as having a crucial impact on CaM/S100A1 binding. The data also suggest that the interaction is calcium-dependent. We also performed competition assays, which suggested that CaM and S100A1 are able to compete for the same binding sites within the TRPM3 N terminus. This is the first time that such an interaction has been shown for TRP family members.

• MeSH
• kalmodulin chemie   genetika   metabolismus   MeSH
• kationtové kanály TRPM chemie   genetika   metabolismus   MeSH
• lidé MeSH
• missense mutace MeSH
• proteiny S100 chemie   genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kalmodulin MeSH
• kationtové kanály TRPM MeSH
• proteiny S100 MeSH
• S100A1 protein MeSH Prohlížeč
• TRPM3 protein, human MeSH Prohlížeč

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
• akademie a ústavy dějiny   MeSH
• dějiny 20. století MeSH
• mikrobiální genetika dějiny   MeSH
• molekulární biologie dějiny   MeSH
• Check Tag
• dějiny 20. století MeSH
• Publikační typ
• časopisecké články MeSH
• historické články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Geografické názvy
• Česká republika 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
• aminokyselinové motivy MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• bakteriální toxiny chemie   genetika   metabolismus   MeSH
• gramnegativní bakterie chemie   genetika   metabolismus   MeSH
• multigenová rodina * MeSH
• regulace genové exprese u bakterií MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bakteriální toxiny 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
• krystalizace MeSH
• krystalografie rentgenová MeSH
• lipoproteiny chemie   MeSH
• Neisseria meningitidis chemie   MeSH
• proteiny vnější bakteriální membrány chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lipoproteiny MeSH
• proteiny vnější bakteriální membrány MeSH