CAS4_PYRCJ
ID CAS4_PYRCJ Reviewed; 213 AA.
AC A3MTK6;
DT 26-NOV-2014, integrated into UniProtKB/Swiss-Prot.
DT 03-APR-2007, sequence version 1.
DT 03-AUG-2022, entry version 69.
DE RecName: Full=CRISPR-associated exonuclease Cas4;
DE EC=3.1.12.1 {ECO:0000269|PubMed:25200083};
GN Name=cas4; OrderedLocusNames=Pcal_0546;
OS Pyrobaculum calidifontis (strain DSM 21063 / JCM 11548 / VA1).
OC Archaea; Crenarchaeota; Thermoprotei; Thermoproteales; Thermoproteaceae;
OC Pyrobaculum.
OX NCBI_TaxID=410359;
RN [1]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=DSM 21063 / JCM 11548 / VA1;
RG US DOE Joint Genome Institute;
RA Copeland A., Lucas S., Lapidus A., Barry K., Glavina del Rio T., Dalin E.,
RA Tice H., Pitluck S., Chain P., Malfatti S., Shin M., Vergez L., Schmutz J.,
RA Larimer F., Land M., Hauser L., Kyrpides N., Mikhailova N., Cozen A.E.,
RA Fitz-Gibbon S.T., House C.H., Saltikov C., Lowe T.M., Richardson P.;
RT "Complete sequence of Pyrobaculum calidifontis JCM 11548.";
RL Submitted (FEB-2007) to the EMBL/GenBank/DDBJ databases.
RN [2]
RP FUNCTION, AND COFACTOR.
RX PubMed=24171432; DOI=10.1021/ja408729b;
RA Lemak S., Beloglazova N., Nocek B., Skarina T., Flick R., Brown G.,
RA Popovic A., Joachimiak A., Savchenko A., Yakunin A.F.;
RT "Toroidal structure and DNA cleavage by the CRISPR-associated [4Fe-4S]
RT cluster containing Cas4 nuclease SSO0001 from Sulfolobus solfataricus.";
RL J. Am. Chem. Soc. 135:17476-17487(2013).
RN [3]
RP X-RAY CRYSTALLOGRAPHY (2.65 ANGSTROMS) IN COMPLEX WITH IRON-SULFUR
RP (2FE-2S), FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES,
RP COFACTOR, SUBUNIT, AND MUTAGENESIS OF CYS-64; ASP-123; GLU-136; PHE-141;
RP GLN-149; TYR-153; TYR-166; CYS-200; CYS-203 AND CYS-209.
RC STRAIN=DSM 21063 / JCM 11548 / VA1;
RX PubMed=25200083; DOI=10.1093/nar/gku797;
RA Lemak S., Nocek B., Beloglazova N., Skarina T., Flick R., Brown G.,
RA Joachimiak A., Savchenko A., Yakunin A.F.;
RT "The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis
RT contains a [2Fe-2S] cluster: crystal structure and nuclease activity.";
RL Nucleic Acids Res. 42:11144-11155(2014).
CC -!- FUNCTION: CRISPR (clustered regularly interspaced short palindromic
CC repeat) is an adaptive immune system that provides protection against
CC mobile genetic elements (viruses, transposable elements and conjugative
CC plasmids). CRISPR clusters contain sequences complementary to
CC antecedent mobile elements and target invading nucleic acids. CRISPR
CC clusters are transcribed and processed into CRISPR RNA (crRNA). A ssDNA
CC exonuclease that has 5' to 3' activity, yielding 5'-OH and 3'-phosphate
CC groups. Has Mn(2+)-dependent endonuclease activity on circular ssDNA.
CC Can unwind dsDNA; unwinding does not require ATP.
CC {ECO:0000269|PubMed:24171432, ECO:0000269|PubMed:25200083}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=exonucleolytic cleavage in the 5'- to 3'-direction to yield
CC nucleoside 3'-phosphates.; EC=3.1.12.1;
CC Evidence={ECO:0000269|PubMed:25200083};
CC -!- COFACTOR:
CC Name=[2Fe-2S] cluster; Xref=ChEBI:CHEBI:190135;
CC Evidence={ECO:0000269|PubMed:25200083, ECO:0000303|PubMed:24171432};
CC Note=Binds 1 [2Fe-2S] cluster per subunit. Not required for nuclease
CC activity, since mutation of the Cys residues leads to a colorless but
CC active protein. {ECO:0000269|PubMed:25200083,
CC ECO:0000303|PubMed:24171432};
CC -!- COFACTOR:
CC Name=Mn(2+); Xref=ChEBI:CHEBI:29035;
CC Evidence={ECO:0000269|PubMed:25200083};
CC Name=Co(2+); Xref=ChEBI:CHEBI:48828;
CC Evidence={ECO:0000269|PubMed:25200083};
CC Note=Mn(2+) required for ssDNA cleavage activity. Can also utilize
CC Co(2+) and to a lesser extent Mg(2+). {ECO:0000269|PubMed:25200083};
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC pH dependence:
CC Optimum pH is 7-9. {ECO:0000269|PubMed:25200083};
CC Temperature dependence:
CC Optimum temperature is 50-70 degrees Celsius.
CC {ECO:0000269|PubMed:25200083};
CC -!- SUBUNIT: Monomer. {ECO:0000269|PubMed:25200083}.
CC -!- SIMILARITY: Belongs to the CRISPR-associated exonuclease Cas4 family.
CC {ECO:0000305}.
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DR EMBL; CP000561; ABO07973.1; -; Genomic_DNA.
DR PDB; 4R5Q; X-ray; 2.65 A; A=1-213.
DR PDBsum; 4R5Q; -.
DR AlphaFoldDB; A3MTK6; -.
DR SMR; A3MTK6; -.
DR STRING; 410359.Pcal_0546; -.
DR PRIDE; A3MTK6; -.
DR EnsemblBacteria; ABO07973; ABO07973; Pcal_0546.
DR KEGG; pcl:Pcal_0546; -.
DR eggNOG; arCOG00790; Archaea.
DR HOGENOM; CLU_1507438_0_0_2; -.
DR OMA; YCPRLLW; -.
DR BRENDA; 3.1.12.1; 30621.
DR Proteomes; UP000001431; Chromosome.
DR GO; GO:0051537; F:2 iron, 2 sulfur cluster binding; IDA:UniProtKB.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-KW.
DR GO; GO:0045145; F:single-stranded DNA 5'-3' exodeoxyribonuclease activity; IDA:UniProtKB.
DR GO; GO:0000014; F:single-stranded DNA endodeoxyribonuclease activity; IDA:UniProtKB.
DR GO; GO:0051607; P:defense response to virus; IEA:UniProtKB-KW.
DR GO; GO:0032508; P:DNA duplex unwinding; IDA:UniProtKB.
DR Gene3D; 3.90.320.10; -; 1.
DR InterPro; IPR013343; CRISPR-assoc_prot_Cas4.
DR InterPro; IPR022765; Dna2/Cas4_DUF83.
DR InterPro; IPR011604; PDDEXK-like_dom_sf.
DR Pfam; PF01930; Cas_Cas4; 1.
DR TIGRFAMs; TIGR00372; cas4; 1.
PE 1: Evidence at protein level;
KW 2Fe-2S; 3D-structure; Antiviral defense; Exonuclease; Hydrolase; Iron;
KW Iron-sulfur; Manganese; Metal-binding; Nuclease.
FT CHAIN 1..213
FT /note="CRISPR-associated exonuclease Cas4"
FT /id="PRO_0000430935"
FT BINDING 64
FT /ligand="[2Fe-2S] cluster"
FT /ligand_id="ChEBI:CHEBI:190135"
FT /evidence="ECO:0000269|PubMed:25200083"
FT BINDING 94
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /evidence="ECO:0000303|PubMed:25200083"
FT BINDING 123
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /evidence="ECO:0000303|PubMed:25200083"
FT BINDING 136
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /evidence="ECO:0000303|PubMed:25200083"
FT BINDING 137
FT /ligand="Mg(2+)"
FT /ligand_id="ChEBI:CHEBI:18420"
FT /evidence="ECO:0000303|PubMed:25200083"
FT BINDING 200
FT /ligand="[2Fe-2S] cluster"
FT /ligand_id="ChEBI:CHEBI:190135"
FT /evidence="ECO:0000269|PubMed:25200083"
FT BINDING 203
FT /ligand="[2Fe-2S] cluster"
FT /ligand_id="ChEBI:CHEBI:190135"
FT /evidence="ECO:0000269|PubMed:25200083"
FT BINDING 209
FT /ligand="[2Fe-2S] cluster"
FT /ligand_id="ChEBI:CHEBI:190135"
FT /evidence="ECO:0000269|PubMed:25200083"
FT MUTAGEN 64
FT /note="C->A: Probably no 2Fe-2S center assembly, wild-type
FT nuclease and DNA unwinding."
FT /evidence="ECO:0000269|PubMed:25200083"
FT MUTAGEN 123
FT /note="D->A: No exonuclease or endonuclease, reduced DNA
FT unwinding activity."
FT /evidence="ECO:0000269|PubMed:25200083"
FT MUTAGEN 136
FT /note="E->A: No exonuclease or endonuclease, very little
FT DNA unwinding activity."
FT /evidence="ECO:0000269|PubMed:25200083"
FT MUTAGEN 141
FT /note="F->A: Increased endonuclease activity."
FT /evidence="ECO:0000269|PubMed:25200083"
FT MUTAGEN 149
FT /note="Q->A: No exonuclease or endonuclease, very little
FT DNA unwinding activity."
FT /evidence="ECO:0000269|PubMed:25200083"
FT MUTAGEN 153
FT /note="Y->A: No exonuclease or endonuclease, reduced DNA
FT unwinding activity."
FT /evidence="ECO:0000269|PubMed:25200083"
FT MUTAGEN 166
FT /note="Y->A: No exonuclease or endonuclease, reduced DNA
FT unwinding activity."
FT /evidence="ECO:0000269|PubMed:25200083"
FT MUTAGEN 200
FT /note="C->A: Probably no 2Fe-2S center assembly, wild-type
FT nuclease and DNA unwinding."
FT /evidence="ECO:0000269|PubMed:25200083"
FT MUTAGEN 203
FT /note="C->A: Probably no 2Fe-2S center assembly, wild-type
FT nuclease and DNA unwinding."
FT /evidence="ECO:0000269|PubMed:25200083"
FT MUTAGEN 209
FT /note="C->A: Probably no 2Fe-2S center assembly, wild-type
FT nuclease and DNA unwinding."
FT /evidence="ECO:0000269|PubMed:25200083"
FT HELIX 9..11
FT /evidence="ECO:0007829|PDB:4R5Q"
FT HELIX 15..18
FT /evidence="ECO:0007829|PDB:4R5Q"
FT HELIX 24..33
FT /evidence="ECO:0007829|PDB:4R5Q"
FT STRAND 37..40
FT /evidence="ECO:0007829|PDB:4R5Q"
FT STRAND 43..47
FT /evidence="ECO:0007829|PDB:4R5Q"
FT HELIX 55..63
FT /evidence="ECO:0007829|PDB:4R5Q"
FT HELIX 65..74
FT /evidence="ECO:0007829|PDB:4R5Q"
FT STRAND 76..80
FT /evidence="ECO:0007829|PDB:4R5Q"
FT TURN 81..83
FT /evidence="ECO:0007829|PDB:4R5Q"
FT HELIX 84..101
FT /evidence="ECO:0007829|PDB:4R5Q"
FT STRAND 107..115
FT /evidence="ECO:0007829|PDB:4R5Q"
FT STRAND 118..128
FT /evidence="ECO:0007829|PDB:4R5Q"
FT STRAND 131..137
FT /evidence="ECO:0007829|PDB:4R5Q"
FT HELIX 143..157
FT /evidence="ECO:0007829|PDB:4R5Q"
FT STRAND 160..165
FT /evidence="ECO:0007829|PDB:4R5Q"
FT STRAND 170..174
FT /evidence="ECO:0007829|PDB:4R5Q"
FT HELIX 177..180
FT /evidence="ECO:0007829|PDB:4R5Q"
FT HELIX 181..191
FT /evidence="ECO:0007829|PDB:4R5Q"
FT STRAND 200..202
FT /evidence="ECO:0007829|PDB:4R5Q"
FT TURN 204..207
FT /evidence="ECO:0007829|PDB:4R5Q"
FT HELIX 208..211
FT /evidence="ECO:0007829|PDB:4R5Q"
SQ SEQUENCE 213 AA; 24673 MW; BE0AE9B0B6781BD7 CRC64;
MELLSPKPLC SVVNCEDLEK LDHVSALNEL RREQEIFKLL PGIYAHRYDF RRVSPSIIND
FEYCPRLLWV QHKLGLKLLS EKSVVSIIRG RILHERYERL LSQYENVVAE YKVEIGDLVG
VVDLVIKRGG EYIPVEIKTG FSKEAHKTQL QIYISMLKAR FGYLVYRNHV EVVHRNDAAL
DVLKKIREIL SAREAPPAKC NSCIFKPICK NLL
