RNA m~6A去甲基化酶Alkbh5的结构与功能研究
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摘要
6-甲基腺苷(N6-methyladenosine, m6A)是真核生物mRNA和长链非编码RNA中最常见、含量最丰富的一种甲基化修饰,它影响mRNA的剪接、输出、翻译和降解等多种RNA代谢过程,在基因的表达调控中起着重要的作用,广泛参与胚胎发育、细胞凋亡、精子发育以及昼夜节律等多种生命活动。m6A修饰是一种动态可逆的RNA修饰,由甲基转移酶和去甲基化酶共同调控。人源Alkbh5蛋白(human AlkB homolog5, hAlkbh5)是近年来继肥胖量与肥胖相关蛋白(fat mass and obesity associated protein, FTO)之后被发现的第二个mRNA m6A去甲基化酶,在mRNA的加工过程以及小鼠的精子发育等生物过程中都起着重要的调控作用。和FTO一样,Alkbh5是Fe2+和a-酮戊二酸(a-ketoglutarate, α-KG)依赖的非血红素加氧酶,属于AlkB家族成员。但与其它AlkB家族成员的底物特异性不同,Alkbh5只对单链RNA/DNA上的m6A修饰具有去甲基化活力。到目前为止,Alkbh5特异性识别m6A修饰及对单链核酸底物选择性识别的分子机制尚不清楚。本论文从结构生物学角度出发,结合生物化学与分子生物学技术对此进行深入的研究与探讨。
本论文通过筛选大量人源Alkbh5截短蛋白片段,优化原核表达纯化条件,最后得到纯度高且稳定性好的人源Alkbh566-292蛋白,体外去甲基化活力实验证明其具有完全的m6A去甲基化酶活性。采用坐滴或悬滴蒸气扩散法获得了Alkbh566-292蛋白含有柠檬酸和乙酸,以及含有Mn2+, Mn2+和α-KG, Mn2+和NOG (N-oxalylglycine), Mn2+和PDCA (pyridine2,4-dicarboxylate)的五种高分辨率晶体,通过分子置换的方法解析它们的三维空间结构。Alkbh566-292整体结构包括7个α螺旋,9个β折叠和多个无规则卷曲,其活性中心形成一个高度保守的DSBH (double-stranded p-helix fold)结构域。将Alkbh566-292的结构和已知的AlkB家族其它成员的结构进行比对,发现Alkbh566-292的核酸识别盖子的二级结构组成以及空间构象存在明显的差异,表明Alkbh5对底物的特异性识别。结构指导的定点突变和体外酶活实验结果证明此盖子上的四个氨基酸R130、K132、Y139以及Y141是Alkbh5识别和结合m6A的关键氨基酸,它们可能通过氢键、静电作用以及π-π堆积作用特异性结合m6A。结构比对分析还发现Alkbh566-292包含一个独特的二硫键C230-C267,这个二硫键在不同种属的Alkbh5蛋白中都是高度保守的,但是在其它AIkB家族成员中不存在。通过与解析出来的其它AlkB家族蛋白DNA复合物结构比较,发现此二硫键导致AlkBFlip3模体与双链DNA上的非甲基修饰链之间存在严重的空间位阻,阻碍了Alkbh5与双链核酸的结合,因此Alkbh5对双链核酸没有催化活性。凝胶阻滞和体外去甲基化酶活实验结果都证实Alkbh566-292C230S突变体蛋白结合双链DNA的能力更强,并且对含有m6A修饰的双链DNA的去甲基化酶活力也显著增强,表明二硫键C230-C267是Alkbh5特异性识别单链而非双链核酸的结构基础。本论文还检测了4种α-KG类似物(NOG、PDCA、丁二酸以及柠檬酸)对Alkbh5的m6A去甲基化酶活力的抑制作用,结果显示分子量小的抑制剂对Alkbh5的去甲基化酶活抑制能力强于分子量大的抑制剂。同FTO相比,Alkbh5的活性中心空腔体积更小,导致其优先结合分子量小的抑制剂。最后,基于Alkbh566-292表面电势分布图,结合表面正电荷区域上关键氨基酸突变蛋白的去甲基化酶活力实验结果,构建了一个Alkbh5结合ssRNA的模型。
总之,本论文的研究结果在一定程度上为了解Alkbh5选择性识别和结合底物的分子机理提供了结构上的理论依据,为AlkB家族蛋白选择性抑制剂的设计提供了分子基础。
N6-methyladenosine (m6A) is the most ubiquitous and abundant modification present in mRNA and long non-coding RNA across eukaryotes, which has been found to function in various pathways of RNA metabolism including mRNA splicing, nuclear export, translation and degradation and to play a vital role in the regulation of gene expression. The m6A modification is involved in diverse life activities such as embryo development, cell apoptosis, spermatogenesis and circadian clock. As a reversible and dynamic modification, m6A methylation is co-regulated by methyltransferases and demethylases. The human AlkB homolog5(hAlkbh5) is the second mRNA m6A demethylase identified after fat mass and obesity associated protein (FTO) recently, playing important regulatory roles in many biological processes including RNA processes and spermatogenesis in mice. Like FTO, Alkbh5is a Fe2+and α-KG (a-ketoglutarate)-dependent non-heme oxygenase, belonging to the AlkB family. Different from the substrate specificity of other AlkB family members, Alkbh5only displays demethylase activity toward the m6A modification in single-stranded nucleic acids. So far, the molecular mechanisms of the specific m6A recognition and single-stranded substrate selection by Alkbh5remain obscure. To elucidate the opened questions, in-depth studies were conducted in this thesis by means of structural biology combined with biochemistry and molecular biology.
Through extensive screening of Alkbh5truncated variants and optimization for expression and purification conditions, the human Alkbh566-292protein of high purity and stability was obtained in this study. In vitro demethylation activity assay revealed that the human Alkbh566-292retained the full demethylation activity. Five high resolution crystals of the human Alkbh5catalytic core in complex with citrate and acetate, Mn2+, Mn2+and a-KG, Mn2+and N-oxalylglycine (NOG), Mn2+and pyridine2,4-dicarboxylate (PDCA) were acquired by vapor diffusion in hanging or sitting drops. Determined by molecular replacement, the Alkbh566-292structure consists of seven a-helices, nine β-sheets and several random coils, the active site of which is mainly composed of a highly conserved double-stranded β-helix fold. Structural comparison between Alkbh566-292and other AlkB proteins revealed that the nucleotide recognition lid of Alkbh566-292shows distinctive composition of secondary structures and spatial conformation, which likely confers the substrate selectivity characteristic of Alkbh5. The structure-guided mutagenesis and in vitro demethylase assays proved that the four residues R130, K132, Y139and Y141located at this region are vital for Alkbh5substrate recognition and binding, which may selectively bind m6A through hydrogen bonds, electrostatic and π-π stacking interactions. The structural alignment also shows that Alkbh5harbors a unique disulfide bond between C230and C267. The disulfide bond in Alkbh5is highly conserved in many different species but is not shared by other AlkB family members. When the double-stranded DNA from other AlkB family complex structures was modeled onto the catalytic site of Alkbh5, the Flip3motif of Alkbh5sterically clashed with the unmethylated strand, impeding the access of dsDNA and dsRNA to the active site of Alkbh5. Both the electrophoretic mobility shift and in vitro demethylase assays supported the stronger dsDNA binding affinity and the higher dsDNA demethylation activity in the C230S mutant, indicating that this disulfide bond is the structural element that determines the single-stranded preference of Alkbh5. Our work also investigated the inhibition of Alkbh5demethylase activity by four a-KG analogs including NOG, PDCA, succinate and citrate. We found that, in contrast to FTO, Alkbh5has potent binding preference towards smaller molecule inhibitors that is likely caused by the smaller active site cavity of Alkbh5. Finally, based on the electrostatic potential map of Alkbh5, we proposed an ssRNA binding model of Alkbh5according to the results of site-directed mutagenesis of the surface residues together with demethylation assays.
Taken together, our studies provide a structural basis for understanding the selective substrate recognition and binding of Alkbh5and offer a foundation for selective drug design against AlkB members.
本论文通过筛选大量人源Alkbh5截短蛋白片段,优化原核表达纯化条件,最后得到纯度高且稳定性好的人源Alkbh566-292蛋白,体外去甲基化活力实验证明其具有完全的m6A去甲基化酶活性。采用坐滴或悬滴蒸气扩散法获得了Alkbh566-292蛋白含有柠檬酸和乙酸,以及含有Mn2+, Mn2+和α-KG, Mn2+和NOG (N-oxalylglycine), Mn2+和PDCA (pyridine2,4-dicarboxylate)的五种高分辨率晶体,通过分子置换的方法解析它们的三维空间结构。Alkbh566-292整体结构包括7个α螺旋,9个β折叠和多个无规则卷曲,其活性中心形成一个高度保守的DSBH (double-stranded p-helix fold)结构域。将Alkbh566-292的结构和已知的AlkB家族其它成员的结构进行比对,发现Alkbh566-292的核酸识别盖子的二级结构组成以及空间构象存在明显的差异,表明Alkbh5对底物的特异性识别。结构指导的定点突变和体外酶活实验结果证明此盖子上的四个氨基酸R130、K132、Y139以及Y141是Alkbh5识别和结合m6A的关键氨基酸,它们可能通过氢键、静电作用以及π-π堆积作用特异性结合m6A。结构比对分析还发现Alkbh566-292包含一个独特的二硫键C230-C267,这个二硫键在不同种属的Alkbh5蛋白中都是高度保守的,但是在其它AIkB家族成员中不存在。通过与解析出来的其它AlkB家族蛋白DNA复合物结构比较,发现此二硫键导致AlkBFlip3模体与双链DNA上的非甲基修饰链之间存在严重的空间位阻,阻碍了Alkbh5与双链核酸的结合,因此Alkbh5对双链核酸没有催化活性。凝胶阻滞和体外去甲基化酶活实验结果都证实Alkbh566-292C230S突变体蛋白结合双链DNA的能力更强,并且对含有m6A修饰的双链DNA的去甲基化酶活力也显著增强,表明二硫键C230-C267是Alkbh5特异性识别单链而非双链核酸的结构基础。本论文还检测了4种α-KG类似物(NOG、PDCA、丁二酸以及柠檬酸)对Alkbh5的m6A去甲基化酶活力的抑制作用,结果显示分子量小的抑制剂对Alkbh5的去甲基化酶活抑制能力强于分子量大的抑制剂。同FTO相比,Alkbh5的活性中心空腔体积更小,导致其优先结合分子量小的抑制剂。最后,基于Alkbh566-292表面电势分布图,结合表面正电荷区域上关键氨基酸突变蛋白的去甲基化酶活力实验结果,构建了一个Alkbh5结合ssRNA的模型。
总之,本论文的研究结果在一定程度上为了解Alkbh5选择性识别和结合底物的分子机理提供了结构上的理论依据,为AlkB家族蛋白选择性抑制剂的设计提供了分子基础。
N6-methyladenosine (m6A) is the most ubiquitous and abundant modification present in mRNA and long non-coding RNA across eukaryotes, which has been found to function in various pathways of RNA metabolism including mRNA splicing, nuclear export, translation and degradation and to play a vital role in the regulation of gene expression. The m6A modification is involved in diverse life activities such as embryo development, cell apoptosis, spermatogenesis and circadian clock. As a reversible and dynamic modification, m6A methylation is co-regulated by methyltransferases and demethylases. The human AlkB homolog5(hAlkbh5) is the second mRNA m6A demethylase identified after fat mass and obesity associated protein (FTO) recently, playing important regulatory roles in many biological processes including RNA processes and spermatogenesis in mice. Like FTO, Alkbh5is a Fe2+and α-KG (a-ketoglutarate)-dependent non-heme oxygenase, belonging to the AlkB family. Different from the substrate specificity of other AlkB family members, Alkbh5only displays demethylase activity toward the m6A modification in single-stranded nucleic acids. So far, the molecular mechanisms of the specific m6A recognition and single-stranded substrate selection by Alkbh5remain obscure. To elucidate the opened questions, in-depth studies were conducted in this thesis by means of structural biology combined with biochemistry and molecular biology.
Through extensive screening of Alkbh5truncated variants and optimization for expression and purification conditions, the human Alkbh566-292protein of high purity and stability was obtained in this study. In vitro demethylation activity assay revealed that the human Alkbh566-292retained the full demethylation activity. Five high resolution crystals of the human Alkbh5catalytic core in complex with citrate and acetate, Mn2+, Mn2+and a-KG, Mn2+and N-oxalylglycine (NOG), Mn2+and pyridine2,4-dicarboxylate (PDCA) were acquired by vapor diffusion in hanging or sitting drops. Determined by molecular replacement, the Alkbh566-292structure consists of seven a-helices, nine β-sheets and several random coils, the active site of which is mainly composed of a highly conserved double-stranded β-helix fold. Structural comparison between Alkbh566-292and other AlkB proteins revealed that the nucleotide recognition lid of Alkbh566-292shows distinctive composition of secondary structures and spatial conformation, which likely confers the substrate selectivity characteristic of Alkbh5. The structure-guided mutagenesis and in vitro demethylase assays proved that the four residues R130, K132, Y139and Y141located at this region are vital for Alkbh5substrate recognition and binding, which may selectively bind m6A through hydrogen bonds, electrostatic and π-π stacking interactions. The structural alignment also shows that Alkbh5harbors a unique disulfide bond between C230and C267. The disulfide bond in Alkbh5is highly conserved in many different species but is not shared by other AlkB family members. When the double-stranded DNA from other AlkB family complex structures was modeled onto the catalytic site of Alkbh5, the Flip3motif of Alkbh5sterically clashed with the unmethylated strand, impeding the access of dsDNA and dsRNA to the active site of Alkbh5. Both the electrophoretic mobility shift and in vitro demethylase assays supported the stronger dsDNA binding affinity and the higher dsDNA demethylation activity in the C230S mutant, indicating that this disulfide bond is the structural element that determines the single-stranded preference of Alkbh5. Our work also investigated the inhibition of Alkbh5demethylase activity by four a-KG analogs including NOG, PDCA, succinate and citrate. We found that, in contrast to FTO, Alkbh5has potent binding preference towards smaller molecule inhibitors that is likely caused by the smaller active site cavity of Alkbh5. Finally, based on the electrostatic potential map of Alkbh5, we proposed an ssRNA binding model of Alkbh5according to the results of site-directed mutagenesis of the surface residues together with demethylation assays.
Taken together, our studies provide a structural basis for understanding the selective substrate recognition and binding of Alkbh5and offer a foundation for selective drug design against AlkB members.
引文
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