Crystal structure of an archaeal intein-encoded homing endonuclease PI-PfuI.
Inteins possess two different enzymatic activities, self-catalyzed protein splicing and site-specific DNA cleavage. These endonucleases, which are classified as part of the homing endonuclease family, initiate the mobility of their genetic elements into homologous alleles. They recognize long asymmetric nucleotide sequences and cleave both DNA strands in a monomer form. We present here the 2.1 A crystal structure of the archaeal PI-PfuI intein from Pyroccocus furiosus. The structure reveals a unique domain, designated here as the Stirrup domain, which is inserted between the Hint domain and an endonuclease domain. The horseshoe-shaped Hint domain contains a catalytic center for protein splicing, which involves both N and C-terminal residues. The endonuclease domain, which is inserted into the Hint domain, consists of two copies of substructure related by an internal pseudo 2-fold axis. In contrast with the I-CreI homing endonuclease, PI-PfuI possibly has two asymmetric catalytic sites at the center of a putative DNA-binding cleft formed by a pair of four-stranded beta-sheets. DNase I footprinting experiments showed that PI-PfuI covers more than 30 bp of the substrate asymmetrically across the cleavage site. A docking model of the DNA-enzyme complex suggests that the endonuclease domain covers the 20 bp DNA duplex encompassing the cleavage site, whereas the Stirrup domain could make an additional contact with another upstream 10 bp region. For the double-strand break, the two strands in the DNA duplex were cleaved by PI-PfuI with different efficiencies. We suggest that the cleavage of each strand is catalyzed by each of the two non-equivalent active sites.
Ichiyanagi
K
K
Department of Structural Biology, Biomolecular Engineering Research Institute 6-2-3 Furuedai, Suita, Osaka, Japan.
Ishino
Y
Y
Ariyoshi
M
M
Komori
K
K
Morikawa
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004247:DNA / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D007438:Introns / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004247:DNA; D004706:Endodeoxyribonucleases; C411963:PI-PfuI intein; D003850:Deoxyribonuclease I