PilD (XcpA, PA4528) is the type IV prepilin peptidase of Pseudomonas aeruginosa, a 290-residue polytopic inner-membrane protein of the peptidase A24 family. It is bifunctional: it cleaves the short basic leader peptide from type IV prepilins at the cytoplasmic face of the inner membrane (EC 3.4.23.43; A24 peptidases are aspartic proteases with an invariant pair of catalytic Asp residues) and then transfers a methyl group from S-adenosyl-L-methionine to the alpha-amino group of the newly exposed N-terminal phenylalanine. A cytoplasmic domain with four conserved cysteines binds zinc, which is required for methylation but not for cleavage. Its substrates are the major pilin PilA and the minor pilins of the type IV pilus, and the pseudopilins XcpT, XcpU, XcpV and XcpW of the Xcp type II secretion system. PilD is therefore required both for type IV pilus biogenesis and for type II secretion of exoproteins such as elastase, phospholipase C and exotoxin A, which accumulate in the periplasm of pilD mutants. Methylation is not required for pilus assembly.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004190 aspartic-type endopeptidase activity | EXP PMID:8096341 A single bifunctional enzyme, PilD, catalyzes cleavage and N... | ACCEPT | Summary: UniProt EXP from Strom et al. 1993: PilD cleaves the prepilin leader peptide and then N-methylates the mature pilin. Reason: Direct enzymatic evidence for the leader peptidase (EC 3.4.23.43). The aspartic-type mechanism is the defining property of the A24 family, whose invariant Asp pair was shown to be catalytic in Vibrio TcpJ and VcpD (PMID:10625704). No more specific active term exists (GO:0008978 prepilin peptidase activity is obsolete). Core function. Supporting Evidence: PMID:8096341 Here we demonstrate that PilD, in addition to cleaving the amino-terminal leader sequences of prepilin, also catalyzes N-methylation of the amino-terminal phenylalanine of the mature pilin PMID:10625704 In this report we show that the TFPPs represent a novel family of bilobed aspartate proteases that is unlike any other protease. |
| GO:0004190 aspartic-type endopeptidase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from PTN000766410, a Bacteria-level node of PTHR30487 whose sole seed is this protein. PilD in its own WITH/FROM is expected for a seed, not circular. Reason: The seed is sound: purified PilD cleaves the PilA leader peptide in vitro (PMID:23255525, PMID:8096341). Endopeptidase activity on prepilin-like substrates, by an aspartic mechanism whose catalytic Asp residues are invariant across the family (PMID:10625704), is the defining property of A24 prepilin peptidases, so placing it on a Bacteria-level node of prepilin peptidases is appropriate even with one seed. Propagation Review Root cause: NO FAILURE CORE Sources checked: UniProtKB:P22610 · pilD PA4528 (self; sole IBD seed, PseudoCAP IDA PMID:23255525 and UniProt EXP PMID:8096341) SUPPORTS TRANSFER Direct in vitro cleavage by purified enzyme; family-wide catalytic Asp pair. Supporting Evidence: PMID:23255525 led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and S-adenosyl methionine-dependent methylation of the mature pilin PMID:10625704 Corresponding aspartate residues are completely conserved throughout this extensive peptidase family. |
| GO:0004190 aspartic-type endopeptidase activity | IDA PMID:23255525 Cell-free production of integral membrane aspartic acid prot... | ACCEPT | Summary: PseudoCAP IDA from Aly et al. 2013: cell-free synthesised, purified PilD cleaves the PilA signal peptide in liposomes and in detergent. Reason: Direct biochemical assay of the purified enzyme, in a paper framing PilD as an integral membrane aspartic acid protease. Sole PseudoCAP seed of the PTN000766410 aspartic-type endopeptidase IBD; sound. Supporting Evidence: PMID:23255525 led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and S-adenosyl methionine-dependent methylation of the mature pilin |
| GO:0004190 aspartic-type endopeptidase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Combined IEA from InterPro IPR000045 (prepilin type IV endopeptidase) and EC 3.4.23.43. Reason: Consistent with the experimental activity; the EC number is experimentally supported for this protein. Supporting Evidence: file:PSEAE/pilD/pilD-uniprot.txt Reaction=Typically cleaves a -Gly-|-Phe- bond to release an N-terminal, basic peptide of 5-8 residues from type IV prepilin |
| GO:0005886 plasma membrane | EXP PMID:8331069 Cleavage, methylation, and localization of the Pseudomonas a... | ACCEPT | Summary: UniProt EXP from Nunn and Lory 1993, which fractionated wild-type and pilD strains and describes PilD acting in the inner membrane. Reason: PilD is a six-TM polytopic inner-membrane protein; its cytoplasmic membrane location was also shown on first characterisation of xcpA (PMID:1898929). Core location of the enzyme. Supporting Evidence: PMID:8331069 This bifunctional enzyme, acting in the inner membrane, cleaves the leader peptides from precursors of pilins and XcpT-W PMID:1898929 This is consistent with a localization in the cytoplasmic membrane in P. aeruginosa, determined after specific expression of the xcpA gene under control of the T7 phi 10 promoter. |
| GO:0005886 plasma membrane | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from PTN000766410, seeded by PilD, E. coli GspO (P25960) and E. coli PppA (Q46836). Reason: All A24 prepilin peptidases are polytopic inner-membrane enzymes that process their substrates at the membrane; three seeds and this protein's own EXP support the node. Propagation Review Root cause: NO FAILURE CORE Supporting Evidence: PMID:8331069 This bifunctional enzyme, acting in the inner membrane, cleaves the leader peptides from precursors of pilins and XcpT-W |
| GO:0005886 plasma membrane | IDA PMID:9466269 Type II protein secretion by Pseudomonas aeruginosa: genetic... | ACCEPT | Summary: PseudoCAP IDA citing Kagami et al. 1998, a paper on temperature-sensitive xcpT mutants and their xcpR suppressors. A seed of the PTN000766410 plasma membrane IBD. Reason: The claim is correct (PMID:1898929, PMID:8331069). The abstract of PMID:9466269 mentions PilD/XcpA only as the peptidase that cleaves and methylates XcpT-W and reports no PilD localization; full text is not available here. The row is not overruled, but the reference is queried. Supporting Evidence: PMID:1898929 This is consistent with a localization in the cytoplasmic membrane in P. aeruginosa |
| GO:0005886 plasma membrane | IEA GO_REF:0000044 | ACCEPT | Summary: UniProt subcellular-location mapping (cell inner membrane) to plasma membrane. Reason: Same claim as the EXP row; the UniProt location is experimentally supported. Supporting Evidence: file:PSEAE/pilD/pilD-uniprot.txt Cell inner membrane {ECO:0000269|PubMed:8331069} |
| GO:0006480 N-terminal protein amino acid methylation | IDA PMID:23255525 Cell-free production of integral membrane aspartic acid prot... | ACCEPT | Summary: PseudoCAP IDA: purified PilD performs SAM-dependent methylation of the mature pilin N terminus, requiring zinc. Reason: PilD itself catalyses this step (N-methylation of the N-terminal Phe of mature pilins and pseudopilins), so it participates in the process. Core. Supporting Evidence: PMID:23255525 Zinc is required for the N-terminal methylation of the mature pilin, but not for signal peptide cleavage. PMID:8096341 also catalyzes N-methylation of the amino-terminal phenylalanine of the mature pilin, using S-adenosyl-L-methionine as a methyl donor |
| GO:0008170 N-methyltransferase activity | IDA PMID:8096341 A single bifunctional enzyme, PilD, catalyzes cleavage and N... | MODIFY | Summary: PseudoCAP IDA from Strom et al. 1993, the paper showing PilD is the prepilin N-methyltransferase. Reason: Correct but general. PilD transfers a methyl group from SAM to the alpha-amino group of the N-terminal Phe of its protein substrates, which is exactly GO:0071885 N-terminal protein N-methyltransferase activity (an is_a descendant of methyltransferase activity on proteins, using SAM). Proposed replacements: N-terminal protein N-methyltransferase activity Supporting Evidence: PMID:8096341 also catalyzes N-methylation of the amino-terminal phenylalanine of the mature pilin, using S-adenosyl-L-methionine as a methyl donor |
| GO:0009297 pilus assembly | IDA PMID:9668097 Amino acid substitutions in PilD, a bifunctional enzyme of P... | MODIFY | Summary: PseudoCAP IDA from Pepe and Lory 1998, which analysed Gly95/Lys96 variants of PilD for peptidase and methylase activities and for pilus assembly in vivo. Reason: Correct but general: PilD processes type IV prepilins, and the gene already carries GO:0043683 type IV pilus assembly. The pilus-assembly data in this paper come from expressing PilD variants in P. aeruginosa (a mutant analysis, IMP), whereas IDA fits its in vitro enzyme assays; query recorded. Proposed replacements: type IV pilus assembly Supporting Evidence: PMID:9668097 Absence of N-methyltransferase activity did not appear to interfere with the ability of P. aeruginosa to assemble functional pili. |
| GO:0015627 type II protein secretion system complex | IDA PMID:1588814 Protein secretion in Pseudomonas aeruginosa: characterizatio... | MARK AS OVER ANNOTATED | Summary: PseudoCAP part_of T2SS complex citing Bally et al. 1992, which showed that XcpA/PilD processes the Xcp pseudopilins in vivo. Reason: PilD is required for type II secretion because it matures the pseudopilins XcpT-W, but the cited abstract shows processing of Xcp proteins by PilD, not that PilD is a subunit of the envelope-spanning secretion complex. PilD is a maturation enzyme shared with the type IV pilus system, encoded outside the xcp cluster. Comparator check: no other reviewed A24 prepilin peptidase (PulO, OutO, E. coli GspO, Vibrio TcpJ/VvpD) carries GO:0015627 in GOA. The role in type II secretion is real but is better captured as a process; the full text was not seen, so the row is marked rather than removed. Supporting Evidence: PMID:1588814 The N-termini of four of the encoded Xcp proteins display similarity to the N-termini of type IV pili, suggesting that XcpA is involved in the processing of these Xcp proteins. This could indeed be demonstrated in vivo. PMID:8331069 These studies demonstrate that the biogenesis of the apparatus of extracellular protein secretion and that of type IV pili share a requirement for PilD. |
| GO:0016020 membrane | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO membrane from IPR000045 (prepilin type IV endopeptidase). Reason: Correct but general; PilD is an inner-membrane (plasma membrane) protein, which the gene already carries with experimental support. Proposed replacements: plasma membrane Supporting Evidence: PMID:8331069 This bifunctional enzyme, acting in the inner membrane |
| GO:0043683 type IV pilus assembly | IMP PMID:1671384 Multiple roles of the pilus biogenesis protein pilD: involve... | ACCEPT | Summary: PseudoCAP IMP from Strom et al. 1991, which restates that pilB, pilC and pilD are needed for pilin modification and assembly into pili. Reason: Core process. PilD cleaves the prepilin leader peptide, a step without which pilin is not assembled; it does work of the assembly process rather than being its substrate. The primary mutant data are in Nunn et al. 1990 (PMID:1971619), cited by this abstract. Supporting Evidence: PMID:1671384 In Pseudomonas aeruginosa, the genes pilB, pilC, and pilD encode proteins necessary for posttranslational modification and assembly of pilin monomers into pilus organelles |
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Download this section (compressed HTML)Q: Should PilD and other bifunctional or T2SS-associated prepilin peptidases carry involved_in GO:0015628 protein secretion by the type II secretion system, given that pilD mutants fail to secrete Xcp substrates because the pseudopilins are not matured? No A24 family member currently carries the term, which suggests a convention to clarify (maturation of machine components vs participation in secretion).
Q: The obsolete GO:0008978 prepilin peptidase activity once captured the EC 3.4.23.43 reaction; is GO:0004190 plus GO:0071885 the intended replacement for bifunctional prepilin peptidases, and should the methyltransferase half be recorded for A24 family members that lack the conserved cysteine domain?
Experiment: Mutate the conserved Asp pair of P. aeruginosa PilD (equivalent to TcpJ Asp125/Asp189) and test leader-peptide cleavage by purified enzyme, to confirm directly that PilD uses the aspartic mechanism assigned to the family.
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