Ichiyanagi K, Ishino Y, Ariyoshi M, Komori K, Morikawa K
Department of Structural Biology, Biomolecular Engineering Research Institute 6-2-3 Furuedai, Suita, Osaka, Japan.
J Mol Biol. 2000 Jul 21;300(4):889-901. doi: 10.1006/jmbi.2000.3873.
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.
内含肽具有两种不同的酶活性，即自我催化的蛋白质剪接和位点特异性DNA切割。这些内切核酸酶被归类为归巢内切核酸酶家族的一部分，可启动其遗传元件向同源等位基因的移动。它们识别长的不对称核苷酸序列，并以单体形式切割DNA的两条链。我们在此展示了来自嗜热栖热菌的古菌PI-PfuI内含肽的2.1埃晶体结构。该结构揭示了一个独特的结构域，在此称为马镫结构域，它插入在Hint结构域和一个内切核酸酶结构域之间。马蹄形的Hint结构域包含一个蛋白质剪接的催化中心，涉及N端和C端残基。插入到Hint结构域中的内切核酸酶结构域由通过内部假二重轴相关的两个亚结构拷贝组成。与I-CreI归巢内切核酸酶不同，PI-PfuI可能在由一对四链β-折叠形成的假定DNA结合裂隙中心有两个不对称催化位点。DNase I足迹实验表明，PI-PfuI在切割位点不对称地覆盖了超过30个碱基对的底物。DNA-酶复合物的对接模型表明，内切核酸酶结构域覆盖了包含切割位点的20个碱基对的DNA双链，而马镫结构域可能与另一个上游10个碱基对区域进行额外接触。对于双链断裂，DNA双链中的两条链被PI-PfuI以不同效率切割。我们认为每条链的切割是由两个不等价的活性位点之一催化的。
