鲫鱼PKR-like（PKZ）Zα结构域与d（GC）、d（TA）重组质粒的亲和性
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摘要
Zα是能够识别并特异性结合左旋DNA(Z-DNA)的蛋白结构域。鲫鱼PKR-like(PKZ)是首次报道的具有Zα结构域的鱼类蛋白激酶。为深入了解鲫鱼PKR-like(PKZ)Zα的功能,本研究构建替换型P_(Zα1Zα1)的cDNA,并运用PCR定点突变方法构建点突变型(P_(Zα)~(K34A),P_(Zα)~(S35A), P_(Zα)~(R39A) P_(Zα)~(P57A))的cDNA,然后与表达载体连接,转化表达菌BL21(DE3)pLysS并诱导表达。同时,诱导表达野生型P_(Zα1Zα2)。镍柱亲和层析纯化获得突变型融合多肽。
另一方面,以pMD18-T质粒为母本质粒,将含有(GC)_6、d(GC)_(13)、d(TA)_(13)片段的特殊序列插入到pMD18-T质粒中。甲基化抑制实验证实重组质粒中的插入序列d(GC)_6、d(GC)_(13)为“潜在”的Z-DNA。另外,还构建了发夹结构的核酸(d(GC)_3T_4d(GC)_3、d(GC)_6T_4d(GC)_6)和寡聚核苷酸(d(GC)_6、d(GC)_(13))。
凝胶阻滞试验分析3种多肽,即野生型P_(Zα1Zα2)、替换型P_(Zα1Zα1)和4个点突变型(P_(Zα)~(K34A), P_(Zα)~(S35A), P_(Zα)~(R39A) P_(Zα)~(P57A)),分别与重组质粒的亲和性。结果显示:野生型P_(Zα)(Zα1Zα2)和替换型P_((Zα1)2)(Zα1Zα1)能够对3种重组质粒的迁移产生明显的阻滞效应,并且随着多肽浓度的增大,阻滞效应越明显。与野生型P_(Zα)(Zα1Zα2)相比,替换型P_((Zα1)2)(Zα1Zα1)结合重组质粒的能力更强。4种突变体都不能与3种重组质粒结合,暗示鲫鱼PKR-like Zα结构域这4个氨基酸残基在结合核酸分子的过程中非常关键。
凝胶阻滞实验还分析了野生型P_(Zα1Zα2)和替换型P_(Zα1Zα1)分别与发夹结构的核酸、寡聚核苷酸的亲和性,结果显示:野生型P_(Zα1Zα2)和替换型P_(Zα1Zα1)能够与其发生微弱的结合,但其结合能力差异不明显。
Zαdomain can recognise and specially bind to left-handed DNA(Z-DNA).The Carassius auratus PKR-like (CaPKR-like)(PKZ), identifided as the first fish protein kinase, contains Z-DNA binding domain, Za. In order to insight the CaPKR-like Zαmotif, the cDNAs encoded substitution P_((Zα1)2)(Zα1Zα1) and site-mutations (P_(Zα)~(K34A),P_(Zα)~(S35A), P_(Zα)~(R39A) P_(Zα)~(P57A)) constructed by site-directed mutagenesis were linked into experssion vector and transformed into BL21(DE3) pLysS, then induced by IPTG The mutation fusion peptides were obtained by affinity chromatography as wel as wildtype P_(Zα1Zα2).
At the same time, three recombinant plasmids for mimic Z-DNA, containing d(GC)_6 or d(GC)_(13) or d(TA)_(13) insert respectively, were successfully constrcuted as well as hairpins (d(GC)_3T_4d(GC)_3,d(GC)_6T_4d(GC)_6) and oligodeoxynucleotides (d(GC)_6, d(GC)_(13)). The results of methylation inhibition demonstrated that d(GC)_6 and d(GC)_(13) inserts were "latent" Z-DNA. Theses implied that CaPKR-like Za peptide could bind to the inserted fragments d(GC)_6 or d(GC)_(13) within the recombinant plasmids then gave rise to the band shift effection.
The wild type P_(Zα1Zα2) and substitution P_(Zα1Zα1) rather than 4 mutations (P_(Zα)~(K34A), P_(Zα)~(S35A), P_(Zα)~(R39A) P_(Zα)~(P57A)) were capable to bind to the three recombinant plasmids in vitro and the affinity enhanced in the present of increasing amounts of protein. In addition, compared with affinity of wild type P_(Zα), substitution P_(Zα1Zα1) was stronger. 4 mutations (P_(Zα)~(K34A), P_(Zα)~(S35A), P_(Zα)~(R39A), P_(Zα)~(P57A)) had no ability to bind to d(GC) and d(TA) recombinant plasmids by the gel mobility shift assay, this result suggested that the 4 amino acids are important to Za binding to DNA.
The affinity was also detected by the native-PAGE that wild type P_(Zα1Zα2) and substitution P_(Zα1Zα1) binding to hairpins and oligodeoxynucleotides respectively. These peptides were weakly capable to bind to hairpins and oligodeoxynucleotides. No difference was observed between wild type P_(Zα1Zα2) and substitution P_(Zα1Zα1) binding to this kinds of nucleis acids.
另一方面,以pMD18-T质粒为母本质粒,将含有(GC)_6、d(GC)_(13)、d(TA)_(13)片段的特殊序列插入到pMD18-T质粒中。甲基化抑制实验证实重组质粒中的插入序列d(GC)_6、d(GC)_(13)为“潜在”的Z-DNA。另外,还构建了发夹结构的核酸(d(GC)_3T_4d(GC)_3、d(GC)_6T_4d(GC)_6)和寡聚核苷酸(d(GC)_6、d(GC)_(13))。
凝胶阻滞试验分析3种多肽,即野生型P_(Zα1Zα2)、替换型P_(Zα1Zα1)和4个点突变型(P_(Zα)~(K34A), P_(Zα)~(S35A), P_(Zα)~(R39A) P_(Zα)~(P57A)),分别与重组质粒的亲和性。结果显示:野生型P_(Zα)(Zα1Zα2)和替换型P_((Zα1)2)(Zα1Zα1)能够对3种重组质粒的迁移产生明显的阻滞效应,并且随着多肽浓度的增大,阻滞效应越明显。与野生型P_(Zα)(Zα1Zα2)相比,替换型P_((Zα1)2)(Zα1Zα1)结合重组质粒的能力更强。4种突变体都不能与3种重组质粒结合,暗示鲫鱼PKR-like Zα结构域这4个氨基酸残基在结合核酸分子的过程中非常关键。
凝胶阻滞实验还分析了野生型P_(Zα1Zα2)和替换型P_(Zα1Zα1)分别与发夹结构的核酸、寡聚核苷酸的亲和性,结果显示:野生型P_(Zα1Zα2)和替换型P_(Zα1Zα1)能够与其发生微弱的结合,但其结合能力差异不明显。
Zαdomain can recognise and specially bind to left-handed DNA(Z-DNA).The Carassius auratus PKR-like (CaPKR-like)(PKZ), identifided as the first fish protein kinase, contains Z-DNA binding domain, Za. In order to insight the CaPKR-like Zαmotif, the cDNAs encoded substitution P_((Zα1)2)(Zα1Zα1) and site-mutations (P_(Zα)~(K34A),P_(Zα)~(S35A), P_(Zα)~(R39A) P_(Zα)~(P57A)) constructed by site-directed mutagenesis were linked into experssion vector and transformed into BL21(DE3) pLysS, then induced by IPTG The mutation fusion peptides were obtained by affinity chromatography as wel as wildtype P_(Zα1Zα2).
At the same time, three recombinant plasmids for mimic Z-DNA, containing d(GC)_6 or d(GC)_(13) or d(TA)_(13) insert respectively, were successfully constrcuted as well as hairpins (d(GC)_3T_4d(GC)_3,d(GC)_6T_4d(GC)_6) and oligodeoxynucleotides (d(GC)_6, d(GC)_(13)). The results of methylation inhibition demonstrated that d(GC)_6 and d(GC)_(13) inserts were "latent" Z-DNA. Theses implied that CaPKR-like Za peptide could bind to the inserted fragments d(GC)_6 or d(GC)_(13) within the recombinant plasmids then gave rise to the band shift effection.
The wild type P_(Zα1Zα2) and substitution P_(Zα1Zα1) rather than 4 mutations (P_(Zα)~(K34A), P_(Zα)~(S35A), P_(Zα)~(R39A) P_(Zα)~(P57A)) were capable to bind to the three recombinant plasmids in vitro and the affinity enhanced in the present of increasing amounts of protein. In addition, compared with affinity of wild type P_(Zα), substitution P_(Zα1Zα1) was stronger. 4 mutations (P_(Zα)~(K34A), P_(Zα)~(S35A), P_(Zα)~(R39A), P_(Zα)~(P57A)) had no ability to bind to d(GC) and d(TA) recombinant plasmids by the gel mobility shift assay, this result suggested that the 4 amino acids are important to Za binding to DNA.
The affinity was also detected by the native-PAGE that wild type P_(Zα1Zα2) and substitution P_(Zα1Zα1) binding to hairpins and oligodeoxynucleotides respectively. These peptides were weakly capable to bind to hairpins and oligodeoxynucleotides. No difference was observed between wild type P_(Zα1Zα2) and substitution P_(Zα1Zα1) binding to this kinds of nucleis acids.
引文
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[2] Samuel CE. The eIF-2 alpha protein kinases,regulators of translation in eukaryotes from yeasts to humans. J. Biol. Chem., 1993, 268(11):7603-7606
[3] Hanks SK, Hunter T. Protein kinases 6.The eukaryotic protein kinase superfamily: kinase(catalytic) domain structure and classification.E4SE5 J, 1995, 9(8):576-596
[4] Chen JJ, Throop MS, Gehrke L, Kuo I, Pal JK, Brodsky M, London IM. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2a (eIF-2a) kinase of rabbit reticulocytes: Homology to yeast GCN2 protein kinase and human double-stranded RNA dependent eIF-2a kinase. Proc. Natl. Acad. Sci. USA., 1991, 88:7729-7733
[5] Inuzuka T, Yun BG, Ishikawa H, Takahashi S, Hori H, Matts RL, Ishimori K, Morishima I. Identification of Crucial Histidines for Heme Binding in the N-terminal Domain of the Heme-regulated eIF2a Kinase. J. Biol. Chem., 2004,279(8):6778-6782
[6] Rafie-Kolpin M, Chefalo PJ, Hussain Z, Hahn J, Uma S, Matt RL, Chen JJ. Two heme-binding domains of heme-regulated eukaryotic initiation factor-2a kinase. J. Biol.Chem., 2000,275(7):5171-5178
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