A Novel “Clip-and-link” Activity of Repeat in Toxin (RTX) Proteins from Gram-negative Pathogens

Abstract

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 ⑀-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 calciumdependent 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.

Unusual Genetic Organization of a Functional Type I Protein Secretion System in Neisseria meningitidis

“…The meningococcal RTXcontaining exoproteins have recently been shown to belong to a new class of RTX proteins that are capable of calciumdependent autoproteolytic cleavage and cross-linking. FrpC was shown to cleave the peptide bond between Asp 414 and Pro 415 ; the resulting amino-terminal fragment is capable of covalent linkage via its carboxy-terminal aspartate residue to the ε-amino group of an internal lysine residue of another FrpC molecule (24). It is likely that the 175-kDa FrpC-related band is the carboxy-terminal fragment of FrpC reported previously (24).…”

“…FrpC was shown to cleave the peptide bond between Asp 414 and Pro 415 ; the resulting amino-terminal fragment is capable of covalent linkage via its carboxy-terminal aspartate residue to the ε-amino group of an internal lysine residue of another FrpC molecule (24). It is likely that the 175-kDa FrpC-related band is the carboxy-terminal fragment of FrpC reported previously (24). The smaller of the two proteins detected in secreted protein preparations of MC58⌬frpC (as well as wildtype cells) could be a similar cleavage product of FrpC2, since the cleavage site identified previously within FrpC is conserved in the smaller protein.…”

Metal assisted peptide bond hydrolysis: Chemistry, biotechnology and toxicological implications

“…This problem is not shared by a tag cleavage procedure which utilizes the Ca(II)/Mn(II) dependent MIIA domain, which is cleaved within minutes at low and temperatures [87,88]. The disadvantage of this process is that a long stretch of unwanted amino acids derived from the cleavage domain remains at the C-terminus of the final protein product.…”

“…140 amino acid residues is necessarily required for the process [86,87]. Several analogous domains cleaving the Asp-Pro bond are known from other microorganisms, but these are specifically Ca(II) dependent [88,89].…”

Characterization of Member of DUF1888 Protein Family, Self-cleaving and Self-assembling Endopeptidase

“…In both cases, sequence Asp-Pro was essential for autocatalysis, and the reaction was accelerated at low pH. The "clip-and-link" activity was reported for two toxins from Gram-negative pathogens (FrpC of Neisseria meningitidis and ApxIVA of Actinobacillus pleuropneumoniae) containing RTX repeats (39). These proteins can autocleave an Asp-Pro bond in a pH-and calcium-dependent manner and covalently link the generated fragment to itself or to another protein molecule by a newly formed isopeptide Asp-Lys bond.…”