A novel "clip-and-link" activity of repeat in toxin (RTX) proteins from gram-negative pathogens. Covalent protein cross-linking by an Asp-Lys isopeptide bond upon calcium-dependent processing at an Asp-Pro bond.
Clinical isolates of Neisseria meningitidis produce a repeat in toxin (RTX) protein, FrpC, of unknown biological activity. Here we show that physiological concentrations of calcium ions induce a novel type of autocatalytic cleavage of the peptide bond between residues Asp(414) and Pro(415) of FrpC that is insensitive to inhibitors of serine, cysteine, aspartate, and metalloproteases. Moreover, as a result of processing, the newly generated amino-terminal fragment of FrpC can be covalently linked to another protein molecule by a novel type of Asp-Lys isopeptide bond that forms between the carboxyl group of its carboxyl-terminal Asp(414) residue and the epsilon-amino group of an internal lysine of another FrpC molecule. Point substitutions of negatively charged residues possibly involved in calcium binding (D499K, D510A, D521K, and E532A) dramatically reduced the self-processing activity of FrpC. The segment necessary and sufficient for FrpC processing was localized by deletion mutagenesis within residues 400-657, and sequences homologous to this segment were identified in several other RTX proteins. The same type of calcium-dependent processing and cross-linking activity was observed also for the purified ApxIVA protein of Actinobacillus pleuropneumoniae. These results define a protein cleavage and cross-linking module of a new class of RTX proteins of Gram-negative pathogens of man, animals, and plants. In the calcium-rich environments colonized by these bacteria this novel activity is likely to be of biological importance.
Osicka
Radim
R
Institute of Microbiology of the Academy of Sciences of the Czech Republic, Vídeòská 1083, CZ-142 20 Prague 4, Czech Republic.
Procházková
Katerina
K
Sulc
Miroslav
M
Linhartová
Irena
I
Havlícek
Vladimír
V
Sebo
Peter
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002384:Catalysis; D003603:Cytotoxins / Q000737:chemistry* / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000472:pathogenicity*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology
D001426:Bacterial Proteins; D003603:Cytotoxins; D008565:Membrane Proteins; C083530:frpC protein, Neisseria meningitidis; D014107:Tosyllysine Chloromethyl Ketone; D014108:Tosylphenylalanyl Chloromethyl Ketone; D002118:Calcium