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.

Invasive infections by Neisseria meningitidis still belong to the most common causes of epidemic bacterial meningitis. Meningococci colonize the upper respiratory tract of about 10% of healthy asymptomatic carriers. Occasionally, however, the bacteria can penetrate across the mucosal barrier, invade the bloodstream, and pass the blood-brain barrier, causing lifethreatening septicemia and/or meningitis. Many potential virulence factors involved in meningococcal pathogenesis have been studied in substantial detail over the past few decades. These comprise the polysaccharide capsule, adhesins like pili, Opa and Opc proteins, and the iron acquisition systems, respectively (1)(2)(3)(4)(5). In contrast to a number of Gram-negative bacterial pathogens, no proteinaceous exotoxins have yet been implicated in the pathogenesis of invasive meningococcal disease. In 1993, however, Thompson and colleagues (6, 7) discovered in meningococci two homologous secreted proteins, FrpC and FrpA (FrpC-like), that belong to the repeat in toxin (RTX) 1 family of proteins characterized by variable numbers of carboxyl-proximal repetitions of a nonapeptide motif LXGGXG(D/ N)DX. A number of RTX proteins have been shown to be involved in virulence of other Gram-negative genera, such as Actinobacillus, Bordetella, Escherichia, Moraxella, Morganella, Pasteurella, Proteus, and Vibrio (8,9). In a previous study, we have shown that genes encoding FrpC-like proteins of variable size are present in most clinical isolates of N. meningitidis and that sera of a majority of patients who had invasive meningococcal disease contain antibodies specifically recognizing these proteins (10). Although the biological activity of FrpC-like proteins remains unknown, the findings suggested that it might be contributing to meningococcal carriage and/or disease.
We report here that the FrpC protein exhibits a novel type of "clip-and-link" self-processing activity. It is shown that FrpC undergoes a unique calcium-dependent autocatalytic processing at an Asp-Pro peptide bond that is accompanied by formation of high molecular weight oligomeric species of FrpC that contain subunits covalently cross-linked through a new type of isopeptide bond. The same type of calcium-dependent processing activity is demonstrated also for another RTX protein of unknown role in infections by Actinobacillus that also contains the novel protein cleavage and cross-linking module.
Plasmids-The plasmid pTYB2N-FLAGfrpC was used for production of the FrpC protein tagged at its amino terminus with an artificial FLAG peptide (DYKDDDDK) inserted as a double-stranded synthetic oligonucleotide encoding the FLAG epitope (5Ј-TATGGATTATAAA-GATGACGATGACAAATCAACGCG-3Ј annealed with 5Ј-TACGCGTT-GATTTGTCATCGTCATCTTTATAATCCA-3Ј) into the NdeI site of pTYB2frpC (10). The pTYB2frpC⌬rtx construct was derived from pTYB2N-FLAGfrpC and was used for production of the truncated FrpC⌬RTX protein lacking the 967 carboxyl-terminal residues of the RTX domain. FrpC⌬RTX was tagged at the amino terminus by the FLAG peptide and at the carboxyl terminus by a His 6 affinity purification tag. pJFFapxIVA1HisCЈ was used for production of the recombinant carboxyl-terminally His-tailed ApxIVA protein in E. coli (12) and was generously provided by J. Frey from the University of Berne, Berne, Switzerland.
Deletion and Site-directed Mutagenesis-Deletions within the frpC⌬rtx allele were obtained by PCR mutagenesis and/or by using the naturally occurring restriction sites in frpC to introduce in-frame deletions and insertions of suitable synthetic oligonucleotide adaptors. The constructions were called pTYB2frpC⌬rtx⌬X-Y where X and Y stand for the first and the last amino acid residues of the polypeptide segment that was deleted in the corresponding protein construct (e.g. the used X-Y deletions were 1-199, 200 -397, 400 -448, 659 -862, 451-657, 451-562, and 564 -657, respectively). Amino acid substitutions were introduced by site-directed PCR mutagenesis using the proofreading Vent DNA polymerase (New England Biolabs) and suitable pairs of mutagenic PCR primers. The PCR products with introduced substitutions were examined for the absence of other undesired mutations by DNA sequencing and introduced into the pTYB2frpC⌬rtx. Complete sequences and detailed schemes of the plasmid constructs will be provided upon request.
Production and Purification of the FrpC-derived Proteins-The different FrpC-derived constructs were produced in the E. coli strain BL21( DE3) transformed with the appropriate plasmid. Exponential 500-ml cultures grown at 30°C in LB medium with 150 g of ampicillin/ml were induced by isopropyl-␤-D-thiogalactopyranoside (1 mM) for 4 h. The cells were washed twice in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl (TN buffer), 5 mM EDTA, resuspended in TN buffer containing 1 mM EDTA (EDTA buffer), and disrupted by sonication. For protein purification by chitin affinity chromatography, the cell extract was cleared at 20,000 ϫ g for 20 min and loaded on a chitin bead column (New England Biolabs) in the EDTA buffer. After washing, buffered 50 mM dithiothreitol was loaded on the column to promote self-excision of the intein-CBD from the fusion protein during overnight incubation at 4°C. The given FrpC-derived protein was then eluted by restoring buffer flow through the column. In the final step, dithiothreitol was removed by gel filtration on Sephadex G-25 in EDTA buffer. All purification steps were performed at 4°C.
SDS-PAGE and Western Blot Analysis-SDS-PAGE and Western blotting were performed according to standard protocols (13). The proteins were separated by 7.5% SDS-PAGE, transferred to nitrocellulose membranes, probed by the anti-FLAG monoclonal antibody M2 (at a 1:3,000 dilution) or the anti-RTX monoclonal antibody 9D4 (at 1:5,000 dilution), and revealed by peroxidase-conjugated secondary antibody (1:3,000) using the chemiluminescence detection system (ECL, Amersham Biosciences).
Amino-terminal Sequencing-Amino-terminal sequences of proteins electroblotted on polyvinylidene difluoride membranes were determined according to standard protocols using the automated protein sequencing system Procise (Applied Biosystems).
In Vitro Calcium-dependent Processing-The concentrations of free calcium ions were adjusted in the reaction mixtures within the range of 100 nM-9.2 mM by a Ca 2ϩ -EDTA buffering system based on calculations using WEBMAXC 2.10 software. 2 Unless indicated otherwise, processing was initiated by addition of CaCl 2 to adjust free Ca 2ϩ to 2 mM and allowed to proceed for 5 min at 37°C before the reaction was stopped by mixing the sample with SDS-PAGE loading buffer containing 10 mM EDTA to titrate out free calcium ions. In unprocessed controls, calcium addition was omitted, and the purified proteins were incubated in EDTA buffer under otherwise identical conditions. For determination of the pH optimum, acetate buffer was used in the pH range 4.0 -5.5, Bis-Tris Propane buffer was used for pH 6.0 -9.5, and CABS buffer was used for pH 10.5-11.5. The FrpC⌬RTX aliquots were supplemented with 100 mM buffer prior to initiation of processing by addition of calcium. The final pH of each reaction was determined using 100 times larger volumes of the same solutions.
FrpC Processing in N. meningitidis-N. meningitidis strains MC58, ⌬NMB0585 (⌬frpC-like), ⌬NMB1415 (⌬frpC), and the "double mutant" (11) were grown on GCB plates at 37°C in an atmosphere containing 5% CO 2 for 12 h. Bacteria were harvested, washed in RPMI 1640 medium and inoculated to A 600 ϭ 0.02 into fresh medium. Expression of the frpC and frpC-like genes was induced by addition of the iron chelator desferrioxamine to 100 M. Where applicable, 0.4 mM EGTA was added to chelate the calcium ions present in the RPMI 1640 medium (ϳ0.42 mM). After 10 h of growth, the bacteria were harvested by centrifugation, and the secreted proteins were precipitated from culture supernatants with acetone; dissolved in 8 M urea, 50 mM Tris-HCl (pH 8.0), and 150 mM NaCl; separated by SDS-PAGE (acrylamide, 7.5%); and transferred to nitrocellulose membrane. The Frp proteins were detected by Western blots with the anti-RTX monoclonal antibody 9D4 (at 1:3,000 dilution).
Densitometric Analysis-Coomassie-stained 7.5% SDS-polyacrylamide gels were digitalized using the PDI 420oe scanner and the program PDQuest software (PDI Inc.). Images were analyzed using AIDA two-dimensional densitometry software version 2.11 (Raytest). The extent of calcium-induced cleavage was calculated using the equation (U Ϫ P)/U (%) where U is the integrated signal intensity of the unprocessed FrpC⌬RTX band in the negative control and P is the integrated signal intensity of the unprocessed FrpC⌬RTX band remaining in the processed protein sample. The integrated signal intensity of the gel background was subtracted from both values. The experiments were repeated four times, and the given values represent the average Ϯ S.D.
Mass Spectrometric Analysis-Twenty-four individual protein bands per analyzed higher molecular weight protein species were excised from Coomassie R-250-stained SDS-polyacrylamide gels. The gel slices were pooled, chopped, and destained as described elsewhere (14). In-gel protein digestions were carried out with sequencing grade trypsin (Promega, 50 ng/l) overnight at 37°C. The generated peptides were extracted with 40% MeCN, 0.5% acetic acid; dried; and reconstituted into 5% MeCN, 0.5% acetic acid prior to separation on a RPC C2/C18 SC 2.1/10 column (Smart System, Amersham Biosciences) in a linear gradient of MeCN in 0.5% acetic acid. The fractionated peptides were dried and reconstituted in 20 l of 5% MeCN, 0.3% acetic prior to analysis by mass spectrometry.
For esterification, 5 l of the peptide mixture was dried and reconstituted in 20 l of a 4:21 acetylchloride:absolute ethanol mixture. After 3 h of incubation at room temperature the mixture was dried and reconstituted with 10 l of 30% MeCN, 0.3% acetic acid.
Mass spectra were acquired using the MALDI-TOF spectrometer BIFLEX II (Bruker-Daltonics, Bremen, Germany) equipped with a nitrogen laser (337 nm) and gridless delayed extraction ion source. Ion acceleration voltage was 19 kV, and the reflectron voltage was set to 20 kV. Spectra were calibrated externally using the monoisotopic [M ϩ H] ϩ ion of the peptide standard somatostatin (Sigma). A saturated solution of ␣-cyano-4-hydroxycinnamic acid in 50% MeCN, 0.3% acetic acid was used as the MALDI matrix. 1 l of the sample was mixed with 1 l of matrix solution on the target, and the droplet was allowed to dry at ambient temperature. Conventional MALDI mass spectra were correlated with theoretical proteolytic peptide maps. The protonated molecules of selected suspect peptides were subjected to postsource decay (PSD) analyses, and the obtained spectra were manually interpreted.

Calcium Ions Induce a Unique Processing of FrpC at an Asp-Pro Peptide Bond and Formation of Extremely Stable
Higher Molecular Weight Forms of FrpC-While purifying the recombinant FrpC protein, we were unable to separate the full-length FrpC (ϳ198 kDa) from its ϳ150-kDa carboxyl-terminal fragment formed during production in E. coli (10). It was, however, noted that the ϳ150-kDa fragment was more abundant when the chelating agent EDTA was omitted from purification buffers. As shown in Fig. 1, when the purified FrpC protein was incubated for 1 h with 2 mM Ca 2ϩ ions instead of 1 mM EDTA, it was almost completely cleaved to the ϳ150-kDa carboxyl-terminal and ϳ45-kDa amino-terminal fragments. More surprisingly, besides the FrpC fragments, several higher molecular weight forms of FrpC also formed during the calcium-induced processing (Fig. 1B, lanes 2, 4, and 6). These higher molecular weight forms were recognized both by the 9D4 anti-RTX monoclonal antibody recognizing the carboxyl-terminal part of FrpC as well as by the anti-FLAG M2 antibody targeting an amino-terminal FLAG tag in the FrpC construct used. This suggested that upon calcium-induced cleavage, the FrpC protein could form oligomers that were not dissociated under the strongly denaturing conditions of sample preparation for SDS-PAGE. Furthermore neither the calcium-dependent FrpC cleavage activity nor its capacity to form higher molecular weight oligomers could be removed by various rather vigorous purification schemes. These comprised both native and denaturing conditions (e.g. 8 M urea solutions) and different combinations of up to three chromatographic steps, such as affinity purification of His 6 -tagged FrpC on nickel-nitrilotriacetic acidagarose and/or chitin affinity purification of the intein-CBDtagged FrpC, ion-exchange, and hydrophobic chromatographies, respectively (data not shown). This strongly suggested that the calcium-dependent cleavage and higher molecular weight formation activities were catalyzed by the FrpC protein itself and not by a contaminating E. coli component. The latter possibility was rather unlikely since no calcium-dependent protein processing activity has been reported for E. coli.
To characterize this activity of FrpC, the amino-terminal sequence was determined for the newly generated FrpC fragment resulting from calcium-induced processing and compared with the sequence of the co-purifying ϳ150-kDa FrpC fragment formed in E. coli that was incubated in 1 mM EDTA. Unexpectedly, while the co-purifying fragment exhibited amino-terminal sequence FAPWVKE corresponding to cleavage between residues Cys 395 and Phe 396 , a single and different amino-terminal sequence of PLALDL was found for the ϳ150-kDa fragment generated by in vitro processing induced by 2 mM calcium ions. Hence both the full-length FrpC and its co-purifying fragment were processed at a novel site between residues Asp 414 and Pro 415 . This cleavage, however, shortened the co-purifying fragment (starting at Phe 396 , molecular mass ϭ 153.5 kDa) by only 2.3 kDa to the processed fragment (starting at Asp 414 , molecular mass ϭ 151.2 kDa), and the two different fragments could not be resolved by SDS-PAGE.
To facilitate a more detailed analysis of processing and oligomerization of FrpC, a truncated FrpC⌬RTX construct, lacking the entire RTX carboxyl-terminal domain and consisting of only the first 862 residues of FrpC, was prepared. It was further fused to a self-excisable chitin affinity tag to allow purification on chitin beads. As documented in Fig. 2, again a carboxyl-terminal fragment (ϳ50 kDa) with the amino-terminal sequence FAPWVKETK was co-purified with FrpC⌬RTX (ϳ95 kDa). As further shown in Fig. 2, in the presence of 2 mM Ca 2ϩ the FrpC⌬RTX protein was also fully competent for processing and oligomerization, yielding an expected ϳ45-kDa amino-terminal fragment, several higher molecular weight forms, and a newly generated carboxyl-terminal fragment exhibiting the expected amino-terminal sequence of PLALDLDGDG. This latter fragment could now be resolved by SDS-PAGE from the co-purifying fragment of FrpC⌬RTX (starting at Phe 396 ), and the two fragments could be quantified separately. Moreover the size of the higher molecular mass products of FrpC⌬RTX generated during processing also fell into a range allowing their better separation by SDS-PAGE (115-200 kDa). The FrpC⌬RTX construct was, therefore, further used for detailed characterization of the calcium-induced processing of FrpC.
Substitutions of the Asp 414 and Pro 415 Residues at the Processing Site Block the Calcium-induced Cleavage and Oligomerization of FrpC-To analyze the sequence specificity of FrpC FIG. 1. Calcium-dependent cleavage of FrpC. A, a schematic representation of FrpC with the FLAG peptide (DYKDDDDK) introduced at its amino terminus and of the non-repetitive portion of FrpC, FrpC⌬RTX, corresponding to residues 1-862 with amino-terminal FLAG tag and carboxyl-terminal His 6 tag. Two identified cleavage sites within FrpC are indicated by arrows. B, the recombinant FrpC protein was purified by a combination of affinity and ion-exchange chromatography as described previously (10) and incubated for 1 h at 37°C in the absence of free calcium ions (Ϫ) with 1 mM EDTA or in the presence of 2 mM free calcium ions (ϩ). The samples were separated by SDS-PAGE (7.5%) and stained with Coomassie Blue or transferred to a polyvinylidene difluoride membrane and immunodetected by either a monoclonal antibody recognizing the carboxyl-proximal RTX repeats (9D4) or the antibody directed against the amino-terminal FLAG tag (M2). HMW forms, high molecular weight forms of FrpC; FrpC, the fulllength 198-kDa FrpC; FrpC-C ter , carboxyl-terminal fragment of FrpC; FrpC-N ter , amino-terminal fragment of FrpC.
processing, three individual substitutions disrupting the Asp-Pro cleavage site were constructed. Asp 414 was replaced by alanine (D414A) or glutamate (D414E) residues, and Pro 415 was substituted by alanine (P415A). As shown in Fig. 3, the substitutions of both Asp 414 and Pro 415 residues by an alanine abolished the calcium-dependent processing, while only a moderate reduction in the rate of FrpC⌬RTX processing was observed upon substitution of Asp 414 by a Glu residue. Furthermore production of higher molecular weight forms could be observed also with the D414E variant. This shows that the processing activity of FrpC requires the presence of an acidic residue at position 414.
Characteristics of the Calcium-dependent Processing Reaction-To further analyze the calcium-dependent cleavage of FrpC, the kinetics, pH and temperature dependence, and the selectivity of the processing activity for divalent cations were determined. The processing products of FrpC⌬RTX formed under various conditions were separated by SDS-PAGE and quantified by densitometric analysis of Coomassie-stained gels. As shown in Fig. 4, addition of 2 mM free calcium ions resulted in rapid FrpC⌬RTX processing with a reaction half-time of about 5 min under the given conditions, and maximal conversion of about 85% was reached within 30 min. The temperature optimum of the processing reaction ranged between 35°C and 45°C (Fig. 5A), and a rather broad optimal pH range between 5.5 and 8.5 was found (Fig. 5B). The calcium dependence of FrpC processing was further studied using a series of free calcium ion concentrations adjusted between 100 nM and 9.2 mM by a Ca 2ϩ -EDTA buffering system (WEBMAXC 2.10). 2 As shown in Fig. 5C, cleavage of FrpC⌬RTX was induced by concentrations of free Ca 2ϩ ions higher than 100 M, and the processing rate continued to increase with rising Ca 2ϩ concentrations up to the yield of about 80% FrpC⌬RTX conversion in 5 min at 9.2 mM Ca 2ϩ . As further shown in Fig. 5D, other divalent cations, such as Zn 2ϩ , Co 2ϩ , Mn 2ϩ , Ni 2ϩ , Mg 2ϩ , or Cu 2ϩ of which some can be found as essential active site ions of metalloproteases, did not significantly influence the cleavage of FrpC. Among the seven tested divalent cations only cadmium could replace calcium and induced FrpC⌬RTX processing at about half the rate induced by equal Ca 2ϩ concentrations (Fig. 5D).
The Processing Activity of FrpC Resides within the Segment Delimited by Residues 400 -657-To identify the domain of FrpC accounting for the Asp 414 -Pro 415 bond cleavage, a set of further truncated constructs was derived from FrpC⌬RTX ( Fig.  7A) and examined for processing activity. As shown in Fig. 7B, neither large deletions within the 397 amino-terminal residues of FrpC nor removal of the 204 carboxyl-terminal residues of FrpC⌬RTX had any effect on the calcium-dependent processing activity. This was, however, ablated by deletion of the segment between residues 451 and 657 and/or by two shorter deletions within this segment comprising residues 451-562 and 564 -657, respectively (Fig. 7). As expected, deletion of the processing site between residues 400 and 448 also abolished FrpC⌬RTX processing. The minimal FrpC segment accounting for the calcium-dependent processing of the Asp 414 -Pro 415 bond was, thereby, delimited by residues 400 -657.

Point Substitutions of Residues Potentially Involved in Binding of Calcium Ions Abolish FrpC Processing-Extensive
screening of the self-processing segment of FrpC (residues 400 -657) for the consensus sequence motifs of a wide range of known proteases and proteins undergoing autocatalytic cleavage failed to yield any useful hits (Network Protein Sequence @nalysis, Institut de Biologie et Chimie des Protéines, Lyon, France). Since histidine residues are frequently involved in catalysis of peptide bond cleavage by other proteases, we examined whether any of the five histidine residues comprised within the processing segment of FrpC could play a role in its activity. However, individual substitutions of these histidine residues had no (H411L, H588A, and H651L) or very minor effect (H440V and H490L) on the rate of the calcium-induced processing as documented in Fig. 8.
In contrast to the missing protease signature motifs, however, the sequence comparisons of the processing segment revealed the presence of five potential calcium binding sequences, which exhibited various degrees of homology to the well defined EF-hand motif (Table I). Of these sequences, two exhibited a particularly high homology (residues 499 -511 and 521-533) and had conserved all the key residues involved in calcium ion coordination to the EF-hand consensus sites. Four of these residues were, therefore, individually replaced. As further shown in Fig. 8, single substitution of the Asp 499 residue by the oppositely charged lysine residue (D499K) and/or substitutions of the Asp 510 and Glu 532 residues by the neutral alanine residues (D510A and E532A) significantly reduced the rate of FrpC⌬RTX processing. The substitution of Asp 521 by a lysine residue (D521K) or deletion of the Asp 510 residue (⌬D510) reduced the processing capacity of FrpC⌬RTX by about 3 orders of magnitude as determined by quantification of FrpC⌬RTXD521K and FrpC⌬RTX⌬D510 processing after 600 min of incubation (data not shown). Complete loss of the activity then resulted from the deletion of Ala 511 in combination with a substitution of Asp 462 by a lysine residue within yet another predicted EF-hand (D462K ϩ ⌬A511). These results demonstrate that residues Asp 462 , Asp 499 , Asp 510 , Asp 521 , and Glu 532 play a crucial role in the processing activity of the protein most likely by being involved in binding the activating calcium ion(s).

Higher Molecular Weight Forms Consist of FrpC Subunits Covalently Cross-linked by a Novel Type of Asp-Lys Isopeptide
Bond-The unusual higher molecular weight oligomers of FrpC that formed during the calcium-induced processing reaction were extremely stable and could not be dissociated by harsh denaturing treatment, such as incubation for 15 min at 100°C in the presence of 8 M urea, 2% SDS, 100 mM dithiothreitol and subsequent separation by SDS-PAGE in gels containing 8 M urea (data not shown). This suggested that in the course of the processing reaction the FrpC subunits were covalently linked. Therefore, the higher molecular weight forms generated by FrpC⌬RTX were examined by mass spectrometry for the presence of peptide cross-links. The two most abundant higher molecular weight forms (indicated in Fig. 2 as Band 1 and Band 2, respectively) were separated by SDS-PAGE and proteolyzed to fragments by trypsin. The obtained peptide mixtures were fractionated by reverse phase chromatography and analyzed by MALDI-TOF mass spectrometry. As illustrated in Fig. 9 by two examples of the numerous acquired spectra, the digests of one of the higher molecular weight forms (Band 1) contained an unpredicted tryptic fragment detected as a protonated species at m/z 1563.7 (Fig. 9A). This was absent in the spectra of tryptic digests of monomeric (unprocessed) FrpC⌬RTX, and its mass did not correspond to any peptide predicted to form upon complete and/or partial digestion of FrpC⌬RTX. Similarly the trypsin digests of the other excised higher molecular weight form of FrpC⌬RTX (Band 2) contained another unpredicted ion species detected at m/z 1311.5 (Fig. 9B). Covalent structures of these suspect peptidic species could be derived from inspection of their respective daughter ion spectra obtained by PSD fragmentation and from examination of the masses of the respective immonium ions (Fig. 9). The identity and structure of the unpredicted tryptic fragments was further corroborated by analysis of PSD spectra of their ethylester derivatives, which confirmed the expected number of free carboxyl groups and fully supported the structures given in Fig. 9. These analyses revealed that upon calcium-induced processing at the Asp 414 -Pro 415 bond, the newly released carboxyl-terminal Asp 414 residue of the amino-terminal FrpC⌬RTX fragment can form a novel isopeptide bond with free ⑀-amino groups of internal lysine residues of another FrpC molecule (or its fragment), yielding formation of the cross-linked higher molecular weight protein forms. Two different lysine residues, Lys 663 and Lys 703 (numbering refers to full-length FrpC sequence), were found engaged in isopeptide bond links to the carboxyl-terminal Asp 414 residue of the amino-terminal fragment of FrpC⌬RTX. Moreover, when the cross-links formed by the further truncated construct FrpC⌬1-299⌬659 -1829 (consisting of residues 300 -658) were analyzed, another two lysine residues, Lys 356 and Lys 405 , respectively, were found engaged in the isopeptide bond with Asp 414 . 3 This suggests a promiscuity of the Asp-Lys isopeptide bond formation in that several different lysine residues of FrpC may be involved in formation of the cross-links within the higher molecular weight FrpC oligomers.

The FrpC and FrpC-like Proteins Secreted by Meningococci Are Processed and Cross-linked in the Presence of Calcium
Ions-It was important to verify whether processing of FrpC and of its FrpC-like variant (initially called FrpA) and formation of their higher molecular weight forms also occurred during secretion by meningococci. This was indeed the case as documented in Fig. 10. Full-length forms of the FrpC  a Putative calcium binding sequences of FrpC with different degrees of homology to the EF-hand consensus motif. Amino acid residues allowed in the EF-hand consensus sequence are given in uppercase letters, and the residues ''not allowed'' are in lowercase letters. Residues at positions 1, 3, 5, 7, 9, and 12, which are typically involved in coordination of calcium ions within well characterized EF-hand binding sites, are printed in bold.
b Positions of putative calcium binding sequences correspond to amino acid residues of FrpC from the FAM20 strain (6).
c Percentages of similarity of putative calcium binding sequences of FrpC to the EF-hand consensus sequence. and/or FrpC-like proteins were strongly detectable only in the culture supernatants of meningococci grown in RPMI 1640 medium supplemented with 0.4 mM EGTA that chelated free calcium ions, while the processed forms of the FrpC and FrpC-like proteins and their respective higher molecular weight forms were largely predominant in supernatants of cultures without added EGTA in which the secreted FrpC was exposed to calcium present in the phosphate-buffered  Fig. 2) formed by FrpC⌬RTX in the presence of calcium ions. The tryptic fragment not matching any of the masses in the theoretical FrpC digest was detected in one of the peptide fractions at m/z 1563.7, and its structure was determined from the PSD daughter ion spectra. The immonium ions of the corresponding amino acid residues are indicated for Lys(m/z 101) and Arg(m/z 129), respectively. B, structure of a different Asp 414 -Lys 663 isopeptide cross-link within another higher molecular weight species of FrpC⌬RTX (Band 2, Fig. 2) that was determined by analysis of daughter ion PSD spectra of an unpredicted tryptic fragment detected at m/z 1311.5. The immonium ions for amino acid residues detected in PSD spectra of m/z 1311.5 are as follows: Val(m/z 72), Asp(m/z 88), Lys(m/z 101), His(m/z 110), Tyr(m/z 136), and Arg(m/z 129). Masses of fragments that lost the methanesulfenic acid (Ϫ64 Da) from oxidized methionine are given in parentheses (21). The residue numbering corresponds that of the full-length sequence of the FrpC variant from N. meningitidis FAM20 (GenBank TM accession number 1706913). Intens., intensity.
RPMI 1640 medium (ϳ0.42 mM). These results strongly suggest that FrpC is also processed and cross-linked when meningococci grow in the calcium-rich environments of the mucosal secretions of nasopharynx and/or in the plasma and liquor during invasive infections.
Homologous Autoprocessing Modules Are Also Functional in Other Bacterial RTX Proteins-Data base searches for sequences homologous to the self-processing module of FrpC (residues 400 -657) revealed the presence of highly similar protein segments (with BLAST E-values lower than 10 Ϫ20 ) in the ApxIVA protein of the animal pathogen Actinobacillus pleuropneumoniae, in several putative proteins of the plant pathogens Xylella fastidiosa and Ralstonia solanacearum, and in the rhizobiocin RzcA of the nitrogen-fixing plant symbiont Rhizobium leguminosarum, respectively (Fig. 11). Like FrpC, all these proteins belong to the RTX family. The capacity of the FrpC homologues to undergo calcium-dependent processing at an Asp-Pro bond was therefore examined for the recombinant ApxIVA protein that was produced in E. coli and purified from inclusion bodies by a combination of ion-exchange and affinity chromatographies. As shown in Fig. 12, in the presence of calcium ions the full-length ApxIVA was processed to the ϳ71-kDa amino-terminal and ϳ131-kDa carboxyl-terminal fragments predicted to form upon cleavage of the Asp 638 -Pro 639 bond, and the amino-terminal amino acid sequence of PLALDL was indeed obtained for the carboxyl-terminal ϳ131-kDa ApxIVA fragment. Moreover the processing of ApxIVA was also accompanied by formation of the higher molecular weight ApxIVA oligomers that again resisted the harsh denaturing conditions of SDS-PAGE separation (Fig. 12). These results suggest that processing of both the FrpC and ApxIVA proteins involved the same mechanism. DISCUSSION We show here that certain RTX proteins of Gram-negative pathogens harbor a novel type of a calcium-dependent clip-andlink activity module. This can cleave an Asp-Pro bond and covalently link the generated fragment to itself or to another protein molecule by a newly formed isopeptide Asp-Lys bond. Such autoprocessing can yield formation of extremely stable protein dimers and higher oligomers as documented here for the RTX proteins FrpC of N. meningitidis and ApxIVA of A. pleuropneumoniae.
The role of the calcium-dependent cleavage and cross-linking step in the unknown biological function of these RTX proteins remains to be determined. Many proteins are, however, initially synthesized as inactive precursors that are activated by cleavage at one or several peptide bonds. Processing of proteins was shown to occur at bonds formed by various pairs of amino acid residues. However, no endoprotease cleaving an Asp-Pro bond has, to our knowledge, been described so far. It has been known for some time that the peptide bond between Asp and Pro residues is unstable in acidic solutions. Prolonged incubation (24 h) in 70% formic acid is indeed a method of chemical fragmentation of proteins at these bonds (15)(16)(17). The heredescribed calcium-dependent processing of the Asp 414 -Pro 415 bond of FrpC appears, however, to be catalyzed by a novel proteolytic module present in a particular class of RTX proteins. The processing occurs at physiological pH and with reaction rates of about 2 orders of magnitude higher than the uncatalyzed chemical cleavage of the Asp-Pro bond. Specific processing at the Asp-Pro bond was up to now reported only for two eukaryotic proteins, the heavy chain precursor of pre-␣-inhibitor (pro-H3) and the MUC2 mucin. At this stage, it is difficult to decide whether the self-processing at the Asp-Pro bond in pro-H3, MUC2, and FrpC follows the same catalytic mechanism. The primary structures of pro-H3, MUC2, and FrpC are very different even for the portions surrounding the Asp-Pro cleavage site. Moreover pro-H3 and MUC2 do not require calcium ions for processing, and substitution of the Asp residue of the Asp-Pro bond of pro-H3 by a Glu residue completely abolished its processing (18,19), while the same substitution only decreased the rate of FrpC cleavage. Furthermore the pH optimum of the processing reaction was between 3.5 and 4.5 for pro-H3, while the processing of FrpC occurred efficiently within a rather broad physiological pH range from 5.5 to 8.5. On the other hand, both low pH and calcium binding may serve the same purpose of modulating the specific structures of the two proteins to allow processing in a similar way.
The mechanism proposed for the chemical cleavage of the Asp-Pro bond in acidic conditions postulates that the reaction is initiated by a nucleophilic attack of the ␤-carboxyl group of the aspartate on the carbonyl carbon engaged in the imide bond with the proline residue. This is expected to yield an unstable intermediate and disruption of the peptide bond (20). Such cleavage reaction occurs exclusively at the peptide bond be- tween Asp and Pro residues because of the more basic character and the higher degree of imide nitrogen protonation of the proline residue in acidic environments as compared with any other amino acid residue (20). The cleavage has been proposed to yield formation of a reactive anhydride at the newly released carboxyl-terminal Asp residue of the fragment that is rapidly hydrolyzed to yield a carboxyl-terminal Asp residue.
In the case of pro-H3 processing, the formation of an anhydride at the carboxyl-terminal Asp has also been suggested and proposed to account for the formation of a covalent bond linking H3 to the chondroitin sulfate chain of the associated bikunin subunit of pre-␣-inhibitor (19). It is plausible to expect that a reactive Asp anhydride also may be formed during the calciumdependent autocatalytic cleavage of the Asp 414 -Pro 415 bond in FrpC. This assumption would allow the proposal of a possible reaction scheme of processing and cross-linking of FrpC as outlined in Fig. 13. Binding of calcium to the processing module of FrpC would promote a conformational change in the protein that would allow interaction of an as yet uncharacterized residue(s) of FrpC with Pro 415 and promote its protonation at physiological pH. This could promote a nucleophilic attack of the ␤-carboxyl group of Asp 414 on the carbonyl carbon and rupture of the imide bond to Pro 415 . A reactive anhydride at Asp 414 could then form and either hydrolyze or get attacked by adjacent ⑀-amino groups of another FrpC molecule to form a new isopeptide Asp-Lys amide bond, such as those observed here to Lys 663 and Lys 703 residues of FrpC.
This model offers hypotheses that can now be tested experimentally. A question to answer is whether formation of the isopeptide bond between the processed amino-terminal fragment of FrpC and another FrpC molecule is also a catalyzed process or just a consequence of the high chemical reactivity of the putative anhydride at Asp 414 . An anhydride ring freely accessible to water molecules would be expected to hydrolyze before it could react with an amine group of another FrpC molecule. Moreover the isopeptide linking reaction of FrpC appears to be rather selective for free ⑀-amino groups of "itself" albeit accepting various FrpC lysine residues (e.g. Lys 663 and Lys 703 in FrpC⌬RTX or Lys 396 and Lys 405 in FrpC⌬1-299⌬659 -1829, respectively). Indeed the high molecular weight forms of FrpC appear to be formed with comparable efficiency by purified FrpC and by crude FrpC in a cell lysate FIG. 11. Homologues of the FrpC clip-and-link activity module are also found in several other RTX proteins. Alignment of the FrpC segment accounting for the self-processing activity of FrpC (residues 400 -657) with the homologous sequence segments of other RTX proteins from four other Gram-negative pathogenic species. The sequence homologues were identified in protein data bases by BLAST searches, and the sequences were aligned using the Lasergene software (DNASTAR) by the Clustal method. The conserved residues are printed in white within black squares. The numbering corresponds to the residue positions in the sequences of the aligned proteins. FrpC  FAM20, FrpC of N 12. ApxIVA of A. pleuropneumoniae also undergoes the calcium-dependent cleavage at an Asp-Pro bond and formation of SDS-PAGE-resistant higher molecular weight oligomers. The recombinant ApxIVA protein was purified by a combination of ionexchange and affinity chromatography and incubated for 2 h at 37°C in the absence (Ϫ) or in the presence (ϩ) of 2 mM calcium ions. The samples were separated by SDS-PAGE (7.5%) and stained with Coomassie Blue. HMW forms, high molecular weight forms of ApxIVA; ApxIVA, the entire 202-kDa ApxIVA; ApxIVA-C ter , carboxyl-terminal fragment of ApxIVA formed by the calcium-dependent cleavage between residues Asp 638 and Pro 639 ; ApxIVA-N ter , amino-terminal fragment of ApxIVA formed by the Asp 638 -Pro 639 bond cleavage.
containing large amounts of unrelated proteins with free primary amine groups (data not shown) as well as upon secretion by meningococci into the RPMI 1640 culture medium containing rather high concentrations of free amino acids. This speaks in favor of a catalyzed formation of the isopeptide bonds between FrpC molecules. We have repeatedly found FrpC to migrate as a dimer under non-denaturing conditions of gel permeation chromatography and blue native electrophoresis (data not shown), and within FrpC dimers, some free ⑀-amino groups of lysine residues of the substrate FrpC molecule could be in the vicinity of the reactive anhydride as soon as it is generated upon rupture of the Asp-Pro bond. On the other hand, the observed efficacy of the amino-terminal fragment cross-linking to the FrpC molecules was rather low, and upon in vitro processing of the purified FrpC⌬RTX protein, most of the generated amino-terminal fragment was detected free FIG. 13. Proposed mechanism of the calcium-dependent processing of the Asp-Pro bond of FrpC and formation of an Asp-Lys isopeptide bond in covalently cross-linked FrpC molecules. A, schematic depiction of a plausible model of the calcium-dependent processing and cross-linking activity of FrpC. Upon calcium binding FrpC undergoes cleavage at the Asp 414 -Pro 415 bond, and the resulting amino-terminal fragment is linked through its free carboxyl-terminal group to an ⑀-amino group of a lysine residue of another FrpC molecule by an Asp-Lys isopeptide bond. B, schematic representation of the possible chemistry of intramolecular Asp-Pro peptide bond cleavage and intermolecular Asp-Lys isopeptide bond formation as derived from the proposed mechanism of Asp-Pro bond cleavage in acids (20). Upon conformational change induced by calcium binding the ␤-carboxyl group of Asp 414 would initiate a nucleophilic attack on the carbonyl carbon of the imide bond with Pro 415 , resulting in an unstable intermediate and disruption of the peptide bond with subsequent formation of a reactive Asp 414 anhydride. This anhydride can be attacked at the carboxyl carbon of the anhydride ring by the free ⑀-amino group of a lysine residue, leading to formation of an amide (isopeptide) bond with either ␣or ␤-carboxyl of the Asp residue. and not cross-linked. This raises the possibility that the observed self-cross-linking of FrpC is just a "surrogate" reaction occurring in vitro because of the absence of the natural human substrate to which the FrpC fragment could be linked with a higher efficiency.
Alternatively the cross-linking of FrpC may serve to aid formation of oligomeric FrpC nets. Several cross-linked FrpC forms of increasing molecular mass were formed in the processing reaction in vitro and could be detected by SDS-PAGE (see Fig. 2). The largest higher molecular weight forms appeared indeed to consist of more than two cross-linked FrpC molecules/fragments. This would go well with the promiscuity in the choice of the ⑀-amino groups of lysines (Lys 396 , Lys 405 , Lys 663 , and Lys 703 ) to which the amino-terminal FrpC fragment could be linked through the Asp 414 residue.
FrpC processing at the Asp 414 -Pro 415 bond appears to involve a novel proteolytic mechanism since it could not be inhibited by typical inhibitors of serine, cysteine, or aspartate proteases. It was completely inhibited only by the metalchelating agents EDTA and EGTA, which inhibit both the non-metalloenzymes activated by calcium or magnesium ions as well as zinc metalloproteases. FrpC, however, does not appear to be a zinc metalloprotease either since the chelators 1,10-phenanthroline and 8-hydroxyquinoline-5-sulfonic acid, which exhibit a high affinity toward Zn 2ϩ and a low affinity toward Ca 2ϩ , were not able to inhibit the calcium-dependent processing of FrpC. Moreover removal of divalent cations from FrpC by the chelating agents followed by titration with calcium ions led to restoration of the processing activity of FrpC, while titration with zinc ions did not (data not shown), and processing of FrpC was not sensitive to phosphoramidon, which inhibits most microbial metalloproteases.
These results suggest that only calcium ions are required for FrpC processing at the Asp 414 -Pro 415 bond. Binding of calcium ions is likely to promote a conformational change in the FrpC structure that allows the processing reaction to occur. Five potential EF-handlike calcium binding sequences were indeed identified within the FrpC self-processing segment. Two of these sequences that exhibit the highest homology to the consensus EF-hand motif, comprising residues 499 -511 and 521-533, respectively, appear to play a crucial role in calciummediated activation of the autoprocessing capacity of FrpC. This is documented by the debilitating effects of the substitutions of residues Asp 499 , Asp 510 , Asp 521 , and Glu 532 on FrpC processing. The arrangement of the potential EF-hand sites in FrpC appears, however, rather unusual. As outlined in the model proposed in Fig. 14, instead of being separated by a helix-loop-helix structure, which typically separates the calcium binding loops in EF-hand proteins, the two predicted calcium binding loops of FrpC could be separated by a very short segment of only 11 residues.
The present results suggest several directions in which the so far unknown biological function of the FrpC-like proteins could be searched. These proteins are secreted by meningococci during invasive disease and might be modulating the function of some host proteins by catalyzing the cross-linking of the host proteins or by covalently attaching their amino-terminal fragment to the host proteins. Alternatively the FrpC self-processing could serve just to form FrpC nets for some unknown purpose. Meningococci typically colonize the nasopharynx, can invade the bloodstream, and/or cross the blood-brain barrier. In all these niches free calcium ions are abundant. The calciumdependent processing and cross-linking activity of FrpC may therefore be of importance for both the commensal and pathogenic lifestyles of meningococci.
FIG. 14. Possible unconventional arrangement of the putative EF-handlike calcium binding sites of FrpC. A, schematic representation of the arrangement of a pair of canonical EF-hand calcium binding sites. Each EF-hand is represented by two helices that are linked by the binding loop in which calcium ions are coordinated mostly via the side chain carboxyl groups of residues designated here as X, Y, Z, ϪX, ϪY, and ϪZ, respectively. The ϪZ residue usually provides bidentate chelation via the side chain carboxylate of a glutamate or occasionally an aspartate residue. Both EF-hands (EF1 and EF2) are linked by a linker loop, which belongs to the most variable regions of proteins containing EF-hands. B, schematic representation of the unusual arrangement of the two putative EF-hand calcium binding sites (residues 499 -510 and residues 521-532) predicted within the "cleaveand-link" activity segment of FrpC with the depiction of the amino acid residues predicted to be involved in the coordination of calcium ions. The two predicted calcium binding loops would be separated by a segment that is too short to allow formation of the typical helix-loophelix structure separating the calcium binding loops in canonical EFhand proteins.