ID PPEP1_CLOD6 Reviewed; 220 AA.
AC Q183R7;
DT 09-JUL-2014, integrated into UniProtKB/Swiss-Prot.
DT 25-JUL-2006, sequence version 1.
DT 27-MAR-2024, entry version 89.
DE RecName: Full=Pro-Pro endopeptidase {ECO:0000303|PubMed:26522134};
DE Short=PPEP-1 {ECO:0000303|PubMed:26522134};
DE EC=3.4.24.89 {ECO:0000269|PubMed:24623589, ECO:0000269|PubMed:26522134};
DE AltName: Full=Zinc metalloprotease Zmp1 {ECO:0000303|PubMed:24303041};
DE Flags: Precursor;
GN Name=zmp1 {ECO:0000303|PubMed:24303041};
GN Synonyms=ppep-1 {ECO:0000303|PubMed:29794027};
GN OrderedLocusNames=CD630_28300;
OS Clostridioides difficile (strain 630) (Peptoclostridium difficile).
OC Bacteria; Bacillota; Clostridia; Eubacteriales; Peptostreptococcaceae;
OC Clostridioides.
OX NCBI_TaxID=272563;
RN [1]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=630;
RX PubMed=16804543; DOI=10.1038/ng1830;
RA Sebaihia M., Wren B.W., Mullany P., Fairweather N.F., Minton N.,
RA Stabler R., Thomson N.R., Roberts A.P., Cerdeno-Tarraga A.M., Wang H.,
RA Holden M.T.G., Wright A., Churcher C., Quail M.A., Baker S., Bason N.,
RA Brooks K., Chillingworth T., Cronin A., Davis P., Dowd L., Fraser A.,
RA Feltwell T., Hance Z., Holroyd S., Jagels K., Moule S., Mungall K.,
RA Price C., Rabbinowitsch E., Sharp S., Simmonds M., Stevens K., Unwin L.,
RA Whithead S., Dupuy B., Dougan G., Barrell B., Parkhill J.;
RT "The multidrug-resistant human pathogen Clostridium difficile has a highly
RT mobile, mosaic genome.";
RL Nat. Genet. 38:779-786(2006).
RN [2]
RP INDUCTION.
RC STRAIN=630;
RX PubMed=23675309; DOI=10.1371/journal.pgen.1003493;
RA Soutourina O.A., Monot M., Boudry P., Saujet L., Pichon C., Sismeiro O.,
RA Semenova E., Severinov K., Le Bouguenec C., Coppee J.Y., Dupuy B.,
RA Martin-Verstraete I.;
RT "Genome-wide identification of regulatory RNAs in the human pathogen
RT Clostridium difficile.";
RL PLoS Genet. 9:E1003493-E1003493(2013).
RN [3]
RP IDENTIFICATION BY MASS SPECTROMETRY, GENE NAME, FUNCTION, COFACTOR,
RP SUBSTRATE SPECIFICITY, SUBCELLULAR LOCATION, ACTIVE SITE, AND MUTAGENESIS
RP OF GLU-143 AND HIS-146.
RC STRAIN=630;
RX PubMed=24303041; DOI=10.1371/journal.pone.0081306;
RA Cafardi V., Biagini M., Martinelli M., Leuzzi R., Rubino J.T., Cantini F.,
RA Norais N., Scarselli M., Serruto D., Unnikrishnan M.;
RT "Identification of a novel zinc metalloprotease through a global analysis
RT of Clostridium difficile extracellular proteins.";
RL PLoS ONE 8:E81306-E81306(2013).
RN [4]
RP IDENTIFICATION BY MASS SPECTROMETRY, FUNCTION, CATALYTIC ACTIVITY,
RP COFACTOR, SUBSTRATE SPECIFICITY, CLEAVAGE MOTIF, KINETIC PARAMETERS,
RP ACTIVITY REGULATION, AND SUBCELLULAR LOCATION.
RC STRAIN=630;
RX PubMed=24623589; DOI=10.1074/mcp.m113.034728;
RA Hensbergen P.J., Klychnikov O.I., Bakker D., van Winden V.J., Ras N.,
RA Kemp A.C., Cordfunke R.A., Dragan I., Deelder A.M., Kuijper E.J.,
RA Corver J., Drijfhout J.W., van Leeuwen H.C.;
RT "A novel secreted metalloprotease (CD2830) from Clostridium difficile
RT cleaves specific proline sequences in LPXTG cell surface proteins.";
RL Mol. Cell. Proteomics 13:1231-1244(2014).
RN [5]
RP FUNCTION, CATALYTIC ACTIVITY, SUBSTRATE SPECIFICITY, DISRUPTION PHENOTYPE,
RP AND SUBCELLULAR LOCATION.
RC STRAIN=630;
RX PubMed=26522134; DOI=10.1016/j.febslet.2015.10.027;
RA Hensbergen P.J., Klychnikov O.I., Bakker D., Dragan I., Kelly M.L.,
RA Minton N.P., Corver J., Kuijper E.J., Drijfhout J.W., van Leeuwen H.C.;
RT "Clostridium difficile secreted Pro-Pro endopeptidase PPEP-1 (ZMP1/CD2830)
RT modulates adhesion through cleavage of the collagen binding protein
RT CD2831.";
RL FEBS Lett. 589:3952-3958(2015).
RN [6]
RP FUNCTION.
RC STRAIN=630;
RX PubMed=26283789; DOI=10.1074/jbc.m115.665091;
RA Peltier J., Shaw H.A., Couchman E.C., Dawson L.F., Yu L., Choudhary J.S.,
RA Kaever V., Wren B.W., Fairweather N.F.;
RT "Cyclic diGMP regulates production of sortase substrates of Clostridium
RT difficile and their surface exposure through ZmpI protease-mediated
RT cleavage.";
RL J. Biol. Chem. 290:24453-24469(2015).
RN [7]
RP REVIEW.
RX PubMed=28636257; DOI=10.1111/mmi.13736;
RA Corver J., Cordo' V., van Leeuwen H.C., Klychnikov O.I., Hensbergen P.J.;
RT "Covalent attachment and Pro-Pro endopeptidase (PPEP-1)-mediated release of
RT Clostridium difficile cell surface proteins involved in adhesion.";
RL Mol. Microbiol. 105:663-673(2017).
RN [8]
RP SUBUNIT, AND MUTAGENESIS OF 117-GLY--THR-120.
RC STRAIN=630;
RX PubMed=29794027; DOI=10.1074/jbc.ra118.003244;
RA Klychnikov O.I., Shamorkina T.M., Weeks S.D., van Leeuwen H.C., Corver J.,
RA Drijfhout J.W., van Veelen P.A., Sluchanko N.N., Strelkov S.V.,
RA Hensbergen P.J.;
RT "Discovery of a new Pro-Pro endopeptidase, PPEP-2, provides mechanistic
RT insights into the differences in substrate specificity within the PPEP
RT family.";
RL J. Biol. Chem. 293:11154-11165(2018).
RN [9]
RP X-RAY CRYSTALLOGRAPHY (1.25 ANGSTROMS) OF 27-220 OF WILD-TYPE AND MUTANTS
RP ALA-143 AND ALA-143/PHE-178 IN COMPLEXES WITH ZINC AND SUBSTRATE PEPTIDE,
RP ACTIVE SITE, DOMAIN, AND MUTAGENESIS OF GLU-143 AND TYR-178.
RC STRAIN=630;
RX PubMed=26211609; DOI=10.1016/j.str.2015.06.018;
RA Schacherl M., Pichlo C., Neundorf I., Baumann U.;
RT "Structural basis of proline-proline peptide bond specificity of the
RT metalloprotease Zmp1 implicated in motility of Clostridium difficile.";
RL Structure 23:1632-1642(2015).
CC -!- FUNCTION: Zinc-dependent endoprotease with a unique preference for
CC proline residues surrounding the scissile bond. Exhibits a high
CC preference for an asparagine at the P2 position and hydrophobic
CC residues (Val, Ile, Leu) at the P3 position. Efficiently cleaves the
CC LPXTG cell surface proteins CD630_28310 and CD630_32460 at multiple
CC cleavage sites in vivo. Has a role in the regulation of C.difficile
CC adhesion versus motility by cleaving surface adhesion proteins such as
CC the collagen binding protein CD630_28310, and is important for
CC efficient infection. Is also able to cleave fibronectin and fibrinogen
CC in vitro; cleaves at the N-terminus of the beta-chain of fibrinogen.
CC Destabilizes the fibronectin network produced by human fibroblasts.
CC Therefore, may be important in key steps of clostridial pathogenesis by
CC degrading extracellular matrix components associated with the gut
CC epithelial cells. To a lesser extent, IgA1, IgA2, and human HSP 90-
CC beta, but not HSP 90-alpha, are also substrates for the enzyme. Is not
CC active on different collagen types, casein and gelatin.
CC {ECO:0000269|PubMed:24303041, ECO:0000269|PubMed:24623589,
CC ECO:0000269|PubMed:26283789, ECO:0000269|PubMed:26522134}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=The enzyme catalyzes the hydrolytic cleavage of peptide bonds
CC between two proline residues.; EC=3.4.24.89;
CC Evidence={ECO:0000269|PubMed:24623589, ECO:0000269|PubMed:26522134};
CC -!- COFACTOR:
CC Name=Zn(2+); Xref=ChEBI:CHEBI:29105;
CC Evidence={ECO:0000255|PROSITE-ProRule:PRU01339,
CC ECO:0000269|PubMed:24303041, ECO:0000269|PubMed:24623589};
CC Note=Binds 1 zinc ion per subunit. {ECO:0000255|PROSITE-
CC ProRule:PRU01339, ECO:0000269|PubMed:24303041,
CC ECO:0000269|PubMed:24623589, ECO:0000269|PubMed:26211609};
CC -!- ACTIVITY REGULATION: Is inhibited by the chelating agent o-
CC phenanthroline in vitro. {ECO:0000269|PubMed:24623589}.
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=77 uM for synthetic FRET peptide DabcylLys-EVNPPPPD-EdansGlu
CC {ECO:0000269|PubMed:24623589};
CC Vmax=0.08 nmol/sec/ug enzyme with synthetic FRET peptide DabcylLys-
CC EVNPPPPD-EdansGlu as substrate {ECO:0000269|PubMed:24623589};
CC Note=kcat is 19 sec(-1) with synthetic FRET peptide DabcylLys-
CC EVNPPPPD-EdansGlu as substrate. {ECO:0000269|PubMed:24623589};
CC -!- SUBUNIT: Monomer. {ECO:0000269|PubMed:29794027}.
CC -!- SUBCELLULAR LOCATION: Secreted {ECO:0000269|PubMed:24303041,
CC ECO:0000269|PubMed:24623589, ECO:0000269|PubMed:26522134}.
CC -!- INDUCTION: Expression is controlled by a type I c-diGMP riboswitch;
CC elevated levels of c-diGMP repress transcription of zmp1.
CC {ECO:0000269|PubMed:23675309}.
CC -!- DOMAIN: The structure of Zmp1 is similar to anthrax lethal factor (LF)
CC metalloprotease domain IV. The S-loop of Zmp1 undergoes large motions
CC upon substrate binding and seems to exhibit a function analogous to
CC that of LF domain III, thus contributing to sequence specificity and
CC substrate binding. An aliphatic-aromatic network and the diverting loop
CC contribute to Pro-Pro specificity. {ECO:0000269|PubMed:26211609}.
CC -!- DISRUPTION PHENOTYPE: Cells lacking this gene retain CD630_28310 on the
CC cell surface, show higher affinity for collagen type I with attenuated
CC virulence in a hamster model of infection.
CC {ECO:0000269|PubMed:26522134}.
CC -!- SIMILARITY: Belongs to the peptidase M34 family. Pro-Pro endopeptidase
CC subfamily. {ECO:0000305}.
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DR EMBL; AM180355; CAJ69718.1; -; Genomic_DNA.
DR RefSeq; WP_011861706.1; NZ_JAUPES010000033.1.
DR RefSeq; YP_001089343.1; NC_009089.1.
DR PDB; 2N6J; NMR; -; A=27-220.
DR PDB; 5A0P; X-ray; 1.40 A; A/B=27-220.
DR PDB; 5A0R; X-ray; 1.25 A; A/B=27-220.
DR PDB; 5A0S; X-ray; 2.56 A; A/B=27-220.
DR PDB; 5A0X; X-ray; 1.70 A; A/B=27-220.
DR PDB; 5N12; X-ray; 1.38 A; A/B=27-220.
DR PDB; 6R4W; X-ray; 1.39 A; A/B=27-220.
DR PDB; 6R4X; X-ray; 1.83 A; A/B=27-220.
DR PDB; 6R4Y; X-ray; 1.45 A; A/B=27-220.
DR PDB; 6R4Z; X-ray; 1.05 A; A/B=27-220.
DR PDB; 6R50; X-ray; 1.81 A; A/B=27-220.
DR PDB; 6R51; X-ray; 1.94 A; A/B/C=27-220.
DR PDB; 6R52; X-ray; 2.02 A; A/B=24-220.
DR PDB; 6R53; X-ray; 1.80 A; A/B=24-220.
DR PDB; 6R54; X-ray; 1.42 A; A/B=27-220.
DR PDB; 6R55; X-ray; 1.40 A; A/B=27-220.
DR PDB; 6R56; X-ray; 1.77 A; A/B=27-220.
DR PDB; 6R57; X-ray; 1.90 A; A/B=27-220.
DR PDB; 6R58; X-ray; 1.90 A; A/B/C/D=27-220.
DR PDB; 6R59; X-ray; 1.65 A; A/B=27-220.
DR PDB; 6R5A; X-ray; 1.48 A; A/B=24-220.
DR PDB; 6R5B; X-ray; 2.06 A; A/B=27-220.
DR PDB; 6R5C; X-ray; 1.88 A; A/B=27-220.
DR PDBsum; 2N6J; -.
DR PDBsum; 5A0P; -.
DR PDBsum; 5A0R; -.
DR PDBsum; 5A0S; -.
DR PDBsum; 5A0X; -.
DR PDBsum; 5N12; -.
DR PDBsum; 6R4W; -.
DR PDBsum; 6R4X; -.
DR PDBsum; 6R4Y; -.
DR PDBsum; 6R4Z; -.
DR PDBsum; 6R50; -.
DR PDBsum; 6R51; -.
DR PDBsum; 6R52; -.
DR PDBsum; 6R53; -.
DR PDBsum; 6R54; -.
DR PDBsum; 6R55; -.
DR PDBsum; 6R56; -.
DR PDBsum; 6R57; -.
DR PDBsum; 6R58; -.
DR PDBsum; 6R59; -.
DR PDBsum; 6R5A; -.
DR PDBsum; 6R5B; -.
DR PDBsum; 6R5C; -.
DR AlphaFoldDB; Q183R7; -.
DR BMRB; Q183R7; -.
DR SMR; Q183R7; -.
DR STRING; 272563.CD630_28300; -.
DR MEROPS; M34.002; -.
DR DNASU; 4913008; -.
DR EnsemblBacteria; CAJ69718; CAJ69718; CD630_28300.
DR GeneID; 66355238; -.
DR KEGG; cdf:CD630_28300; -.
DR KEGG; pdc:CDIF630_03094; -.
DR PATRIC; fig|272563.120.peg.2981; -.
DR eggNOG; ENOG5030AA8; Bacteria.
DR OrthoDB; 2615003at2; -.
DR BioCyc; PDIF272563:G12WB-2990-MONOMER; -.
DR BRENDA; 3.4.24.89; 1473.
DR Proteomes; UP000001978; Chromosome.
DR GO; GO:0005576; C:extracellular region; IEA:UniProtKB-SubCell.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-KW.
DR GO; GO:0008237; F:metallopeptidase activity; IEA:UniProtKB-KW.
DR GO; GO:0006508; P:proteolysis; IEA:UniProtKB-KW.
DR CDD; cd20183; M34_PPEP; 1.
DR Gene3D; 3.40.390.10; Collagenase (Catalytic Domain); 1.
DR InterPro; IPR014781; Anthrax_toxin_lethal/edema_N/C.
DR InterPro; IPR047568; ATLF-like_dom.
DR InterPro; IPR024079; MetalloPept_cat_dom_sf.
DR Pfam; PF07737; ATLF; 1.
DR SUPFAM; SSF55486; Metalloproteases ('zincins'), catalytic domain; 1.
DR PROSITE; PS51995; ATLF; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Hydrolase; Metal-binding; Metalloprotease; Protease;
KW Reference proteome; Secreted; Signal; Virulence; Zinc.
FT SIGNAL 1..26
FT /evidence="ECO:0000255"
FT CHAIN 27..220
FT /note="Pro-Pro endopeptidase"
FT /id="PRO_0000429768"
FT DOMAIN 35..220
FT /note="ATLF-like"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU01339"
FT REGION 101..103
FT /note="Interacts with substrate peptide"
FT /evidence="ECO:0000269|PubMed:26211609"
FT REGION 117..119
FT /note="Interacts with substrate peptide"
FT /evidence="ECO:0000269|PubMed:26211609"
FT ACT_SITE 143
FT /note="Proton acceptor"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU01339,
FT ECO:0000305|PubMed:24303041, ECO:0000305|PubMed:26211609"
FT BINDING 142
FT /ligand="Zn(2+)"
FT /ligand_id="ChEBI:CHEBI:29105"
FT /ligand_note="catalytic"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU01339,
FT ECO:0000269|PubMed:26211609"
FT BINDING 146
FT /ligand="Zn(2+)"
FT /ligand_id="ChEBI:CHEBI:29105"
FT /ligand_note="catalytic"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU01339,
FT ECO:0000269|PubMed:26211609"
FT BINDING 178
FT /ligand="Zn(2+)"
FT /ligand_id="ChEBI:CHEBI:29105"
FT /ligand_note="catalytic"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU01339"
FT BINDING 185
FT /ligand="Zn(2+)"
FT /ligand_id="ChEBI:CHEBI:29105"
FT /ligand_note="catalytic"
FT /evidence="ECO:0000255|PROSITE-ProRule:PRU01339,
FT ECO:0000269|PubMed:26211609"
FT SITE 135
FT /note="Interacts with substrate peptide"
FT /evidence="ECO:0000269|PubMed:26211609"
FT SITE 142
FT /note="Interacts with substrate peptide"
FT /evidence="ECO:0000269|PubMed:26211609"
FT SITE 178
FT /note="Transition state stabilizer"
FT /evidence="ECO:0000305|PubMed:26211609"
FT MUTAGEN 117..120
FT /note="GGST->SERV: Becomes unable to cleave VNPPVP, nor is
FT able to cleave PLPPVP, the optimal substrate peptide for
FT PPEP-2 from P.alvei."
FT /evidence="ECO:0000269|PubMed:29794027"
FT MUTAGEN 143
FT /note="E->A: Still able to bind zinc. Highly reduced
FT activity on fibronectin. Loss of activity on fibrinogen.
FT Shows a surprising 18% residual proteolytic activity on a
FT substrate peptide. Total loss of proteolytic activity; when
FT associated with F-178."
FT /evidence="ECO:0000269|PubMed:24303041,
FT ECO:0000269|PubMed:26211609"
FT MUTAGEN 146
FT /note="H->A: Not able to bind zinc. Highly reduced activity
FT on fibronectin. Loss of activity on fibrinogen."
FT /evidence="ECO:0000269|PubMed:24303041"
FT MUTAGEN 178
FT /note="Y->F: Shows a surprising 41% residual proteolytic
FT activity on a substrate peptide. Total loss of proteolytic
FT activity; when associated with A-143."
FT /evidence="ECO:0000269|PubMed:26211609"
FT HELIX 28..38
FT /evidence="ECO:0007829|PDB:6R4Z"
FT TURN 39..41
FT /evidence="ECO:0007829|PDB:6R4Z"
FT STRAND 46..48
FT /evidence="ECO:0007829|PDB:5A0R"
FT HELIX 51..61
FT /evidence="ECO:0007829|PDB:6R4Z"
FT HELIX 66..74
FT /evidence="ECO:0007829|PDB:6R4Z"
FT STRAND 79..84
FT /evidence="ECO:0007829|PDB:6R4Z"
FT HELIX 86..88
FT /evidence="ECO:0007829|PDB:6R4Z"
FT HELIX 90..95
FT /evidence="ECO:0007829|PDB:6R4Z"
FT TURN 104..107
FT /evidence="ECO:0007829|PDB:6R53"
FT HELIX 110..112
FT /evidence="ECO:0007829|PDB:6R4Z"
FT STRAND 114..124
FT /evidence="ECO:0007829|PDB:6R4Z"
FT TURN 125..127
FT /evidence="ECO:0007829|PDB:2N6J"
FT STRAND 132..134
FT /evidence="ECO:0007829|PDB:6R4Z"
FT STRAND 136..138
FT /evidence="ECO:0007829|PDB:5N12"
FT HELIX 139..151
FT /evidence="ECO:0007829|PDB:6R4Z"
FT STRAND 153..155
FT /evidence="ECO:0007829|PDB:6R4Z"
FT HELIX 156..158
FT /evidence="ECO:0007829|PDB:6R4Z"
FT HELIX 160..169
FT /evidence="ECO:0007829|PDB:6R4Z"
FT TURN 170..173
FT /evidence="ECO:0007829|PDB:6R4Z"
FT TURN 176..181
FT /evidence="ECO:0007829|PDB:6R4Z"
FT HELIX 183..196
FT /evidence="ECO:0007829|PDB:6R4Z"
FT HELIX 198..207
FT /evidence="ECO:0007829|PDB:6R4Z"
FT HELIX 209..219
FT /evidence="ECO:0007829|PDB:6R4Z"
SQ SEQUENCE 220 AA; 24319 MW; DD38BE785EFF73F4 CRC64;
MRPSKKLLIA IISIFLISSV PVSAHADSTT IQQNKDTLSQ IVVFPTGNYD KNEANAMVNR
LANIDGKYLN ALKQNNLKIK LLSGKLTDEK EYAYLKGVVP KGWEGTGKTW DDVPGLGGST
VALRIGFSNK GKGHDAINLE LHETAHAIDH IVLNDISKSA QFKQIFAKEG RSLGNVNYLG
VYPEEFFAES FAYYYLNQDT NSKLKSACPQ TYSFLQNLAK
//
