Crystal structure of intein homing endonuclease II encoded in DNA polymerase gene from hyperthermophilic Archaeon Thermococcus kodakaraensis strain KOD1
Crystal structure of intein homing endonuclease II encoded in DNA polymerase gene from hyperthermophilic Archaeon Thermococcus kodakaraensis strain KOD1
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DOI：
10.1002/prot.20858
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发表时间：
2006-05-15
影响因子：
2.9
通讯作者：
Kai, Y
中科院分区：
文献类型：
--
作者：
Matsumura, H;Takahashi, H;Kai, Y
导论.内含肽归巢核酸内切酶是一种催化蛋白质剪接和DNA双链切割的双功能蛋白。1-3蛋白质剪接是一个翻译后过程，涉及精确切除一个插入蛋白质结构域，称为内含肽。内含肽通常表现出位点特异性核酸内切酶活性，其识别并切割缺乏其编码DNA序列的DNA序列。识别序列通常是不对称的，长度为12- 40-bp。3在此，我们报道了PI-TkoII的晶体结构，PI-TkoII是来自超嗜热古菌Thermococcus kodakaraensis菌株KOD 1的内含肽内切核酸酶II。PI-TkoII是polA基因的产物：成熟的KOD DNA聚合酶。4迄今为止，内含肽归巢核酸内切酶的晶体结构仅描述了来自酿酒酵母的PI-SceI、来自Xenopi分枝杆菌的细菌促旋酶A亚基中的5，6 GyrA内含肽、7和来自激烈火球菌的PI-PfuI。8 PI-TkoII与其他内含肽具有较低的序列相似性，包括PI-SceI（411个氨基酸重叠中的21.7%同一性）、GyrA内含肽（61个氨基酸重叠中的34.4%同一性）和PI-PfuI（322个氨基酸重叠中的22.7%同一性）。虽然PI-PfuI只报道了古细菌内含肽的结构，但PI-TkoII和PI-PfuI的分子量非常不同，分别为62和53 kDa。PI-TkoII的最小识别序列包括一个16 bp片段（5-CAGCTACTACGGTTAC-3），9与其他内含肽归巢核酸内切酶相比相对较短。PI-TkoII的结构信息为特定核酸内切酶活性的机制提供了新的见解。在本研究中，我们还讨论了PI-TkoII，PI-PfuI和PI-SceI之间的结构域架构的相似性和差异。天然和SeMet PI-TkoII的晶体结构分别以2.7和2.5 μ m分辨率解析（表I）。由于未观察到天然和SeMet PI-TkoII之间存在明显的结构差异，因此我们在此描述了2.5-nm分辨率下SeMet PI-TkoII的晶体结构。最终的模型包括537个残基，279个水分子，6个甘油和8个硫酸盐。根据蛋白质结构分类数据库（SCOP），10 PI-TkoII是“Hedgehog/内含肽（Hint）结构域”折叠的成员。该分子的总大小约为100 65 40 nm，由四个不同的结构域组成[图1（A）]：（内切：残基273-432，绿色），提示结构域（提示：残基1-127和496-537，深蓝色）、结构域III（III：残基433-495，浅蓝色）和结构域IV（IV：残基128-272，粉红色）。结构比较显示，核酸内切酶和Hint结构域的结构主要在PI-TkoII、PI-PfuI和PI-SceI中保守，而结构域III和IV则不保守[图1（A-D）]。结构域III位于缩写：PI-TkoII，来自超嗜热古菌Thermococcus kodakaraensis菌株KOD 1的内含肽核酸内切酶II; PI-SceI，来自S.酿酒酵母;来自激烈火球菌的PI-PfuI内含肽核酸内切酶; MAD，多波长异常色散; SeMet，硒代蛋氨酸。
Introduction. Intein homing endonucleases are bifunctional proteins catalyzing both protein splicing and sitespecific DNA double-strand cleavage. 1–3 Protein splicing is a posttranslational process involving precise excision of an intervening protein domain, termed an intein. An intein often exhibits site-specific endonuclease activity, which recognizes and cleaves the DNA sequence lacking its coding DNA sequence. The recognition sequences are usually asymmetrical and 12-to 40-bp long. 3 Herein, we report the crystal structure of PI-TkoII, an intein endonuclease II from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1. PI-TkoII is a product of the polA gene: mature KOD DNA polymerase. 4 To date, crystal structures of intein homing endonucleases have only been described for PI-SceI from Saccharomyces cerevisiae, 5, 6 GyrA intein in bacterial gyrase A subunit from Mycobacterium xenopi, 7 and PI-PfuI from Pyroccocus furiosus. 8 PI-TkoII shares a low sequence similarity to the other inteins including PI-SceI (21.7% identity in 411 amino acids overlap), GyrA intein (34.4% identity in 61 amino acids overlap), and PI-PfuI (22.7% in 322 amino acids overlap). Although the only structure of archaeal intein has been reported for PI-PfuI, molecular masses are very different between PI-TkoII and PI-PfuI, with 62 and 53 kDa, respectively. The minimal recognition sequence for PI-TkoII involves a 16-bp fragment (5-CAGCTACTACGGTTAC-3), 9 which is relatively short compared with other intein homing endonucleases. Structural information on PI-TkoII provides new insights for mechanisms involved in specific endonuclease activity. In the present study, we also discuss similarities and differences in domain architecture between PI-TkoII, PI-PfuI, and PI-SceI.Results and Discussion. Crystal structures of native and SeMet PI-TkoII were solved at 2.7-and 2.5-Å resolution, respectively (Table I). Because no obvious structural differences between the native and SeMet PI-TkoII were observed, we describe here crystal structure of SeMet PI-TkoII at 2.5-Å resolution. The final model includes 537 residues, 279 water molecules, six glycerols, and eight sulfates. According to the Structural Classification of Proteins database (SCOP), 10 PI-TkoII is a member of the “Hedgehog/intein (Hint) domain” fold. The molecule has an overall size of approximately 100 65 40 Å, and consists of four distinct domains [Fig. 1 (A)]: the endonuclease domain (Endo: residues 273–432, green), the Hint domain (Hint: residues 1–127 and 496–537, dark blue), domain III (III: residues 433–495, light blue), and domain IV (IV: residues 128–272, pink). Structural comparisons showed that structures of endonuclease and Hint domains were mainly conserved among PI-TkoII, PI-PfuI, and PI-SceI, whereas domains III and IV were not [Fig. 1 (A–D)]. Domain III is located atAbbreviations: PI-TkoII, an intein endonuclease II from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1; PI-SceI, intein endonuclease from S. cerevisiae; PI-PfuI intein endonuclease from Pyroccocus furiosus; MAD, multiwavelength anomalous dispersion; SeMet, selenomethionine.