Structure and function of two different threonyl-tRNA synthetases of crenarchaea

两种不同的crenarchaea苏氨酰-tRNA合成酶的结构和功能

基本信息

批准号：
20570104
负责人：
TAKENAKA Akio
金额：
$ 3.08万
依托单位：
Iwaki Meisei University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

项目摘要

Threonyl-tRNA synthetase (ThrRS) plays an essential role in protein synthesis by catalyzing aminoacylation of tRNA^<Thr> and by editing misacylation. ThrRS is generally composed of the three domains, an N-terminal editing domain, a catalytic domain and an anticodon-binding domain. In general, an organism possesses one kind of gene for ThrRS. However, it has been recently found that some organisms have two different genes for ThrRS in the genome, suggesting that their proteins ThrRS-1 and ThrRS-2 function separately and complement each other in threonylation of tRNA^<Thr> ; one for catalysis and the other for editing of misacylated Ser-tRNA^<Thr>. From sequence alignment of ThrRS-1 and ThrRS-2 with those of other organisms, we have found that the editing domain in ThrRS from archaea are different from those in bacteria and eukaryotes. Furthermore, in several creanarchaea including Aeropyrum pernix K1 and Sulfolobus tokodaii strain 7, each contains two genes encoding either the catalytic … More or the editing domains of ThrRS. In order to clarify the structural basis for the evolutionary divergence, the two types of ThrRSs from crenarchaea Aeropyrum pernix and those from Sulfolobus tokodaii (ApThrRS-1, ApThrRS-2, StThrRS-1 and StThrRS-2) have been overexpressed in Eschericha coli, purified and successfully crystallized by the hanging-drop vapor-diffusion method. Diffraction data were collected, and the structure of selenomethionine-labeled ApThrRS-1 crystal has been solved usingthe MAD method, and the atomic parameters were refined by the least-squares method at 2.3 resolution. ApThrRS-1 is a dimeric enzyme composed of two identical subunits, each containing two domains for the catalytic reaction and for anticodon binding. The essential editing domain is completely missing as expected. These structural features reveal that ThrRS-1 catalyzes only the aminoacylation of the cognate tRNA, suggesting the necessity of the second enzyme ThrRS-2 for editing. Since the N-terminal sequence of ApThrRS-2 is similar to the sequence of the editing domain of ThrRS from Pyrococcus abyssi, ApThrRS-2 has been expected to catalyze deaminoacylation of a misacylated serine moiety at the CCA terminus. The tertiary structure of ThrRS-2 was constructed based on the sequence alignment with similar protein, based on which the dimer structure formed between the two editing domains. Gel shift assay experiments shows that ThrRS-1 and ThrRS-2 do not interact to form a dimer and that each forms a self dimer. Furthermore, we have found that tRNA^<Thr> interacts to both, but ThrRS-1 is strongly bound to tRNA^<Thr>. Finally we succeeded to crystallize the complexes between tRNA^<Thr> and ThrRS-1 and between tRNA and ThrRS-2, respectively. X-Ray analyses of these crystals will reveal the interaction geometry from which the specificities of ThrRS-1 and ThrRS-2 to tRNA^<Thr> will be clarified. Less

苏氨酰-tRNA合成酶(ThrRS)通过催化tRNA^<Thr>的氨酰化和编辑三酰化在蛋白质合成中发挥重要作用，ThrRS通常由三个结构域组成：N端编辑结构域、催化结构域和反密码子。一般来说，生物体拥有一种ThrRS基因，但最近发现一些生物体的基因组中具有两种不同的ThrRS基因。蛋白质 ThrRS-1 和 ThrRS-2 在 tRNA^<Thr> 的苏酰化过程中各自发挥作用并相互补充；根据 ThrRS-1 和 ThrRS 的序列比对，其中一种用于催化，另一种用于编辑错误酰基化的 Ser-tRNA^<Thr>。 -2 与其他生物体相比，我们发现古细菌中的 ThrRS 编辑域与细菌和真核生物中的编辑域不同。此外，在包括 Pernix 在内的几种古细菌中。 K1 和 Sulfolobus tokodaii 菌株 7 各自包含两个编码 ThrRS 催化域或编辑域的基因。为了阐明进化差异的结构基础，来自 crenarchaea Aeropyrum pernix 和 Sulfolobus tokodaii 的两种类型的 ThrRS。（ApThrRS-1、ApThrRS-2、StThrRS-1 和 StThrRS-2）在埃希氏菌中过表达采用悬滴蒸气扩散法纯化并成功结晶，收集了硒代蛋氨酸标记的 ApThrRS-1 晶体的结构，并使用 MAD 方法解析了原子参数。 ApThrRS-1 是一种二聚酶，由两个相同的亚基组成，每个亚基包含两个用于催化反应和反密码子结合的结构域。正如预期的那样，必需的编辑域完全缺失。这些结构特征表明 ThrRS-1 只催化同源 tRNA 的氨酰化，这表明需要第二种酶 ThrRS-2 进行编辑，因为 ApThrRS-2 的 N 端序列是与来自深渊火球菌的 ThrRS 编辑域的序列相似，ApThrRS-2 预计可催化三酰化丝氨酸部分的脱氨酰化作用。基于与相似蛋白的序列比对构建了ThrRS-2的CCA末端，基于此，两个编辑域之间形成的二聚体结构表明ThrRS-1和ThrRS-2不相互作用。此外，我们发现 tRNA^<Thr> 与两者相互作用，但 ThrRS-1 与 tRNA^<Thr> 紧密结合。分别结晶 tRNA^<Thr> 和 ThrRS-1 之间以及 tRNA 和 ThrRS-2 之间的复合物，这些晶体的 X 射线分析将揭示 ThrRS-1 和 ThrRS-2 对 tRNA^ 的特异性的相互作用几何结构。 <Thr> 将被澄清。

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Crystallization and preliminary crystallographic studies of putative 3'-terminal phosphate cyclase from the crenarchaeon Sulfolobus tokodaii

来自 crenarchaeon Sulfolobus tokodaii 的假定 3-末端磷酸环化酶的结晶和初步晶体学研究

DOI：
发表时间：
2009
期刊：
影响因子：
0
作者：
S.Shimuzu; T.Sekiguchi; A.Takenaka
通讯作者：
A.Takenaka

X-Ray Analyses of Two Evolutionary Different Threonyl-tRNA Synthetases Which Prefer a Functionby Supplementing Their Defects to Each Other in Crenarchaea

Crenarchaea 中两种进化不同的苏氨酰-tRNA 合成酶的 X 射线分析，它们通过相互补充缺陷来偏好功能

DOI：
发表时间：
2009
期刊：
影响因子：
0
作者：
S.Shimizu; Y.Sato; E.C.M.Juan; Y.Miyashita; T.Sagara; K.Suzuki; M.Tsunoda; T.Sekiguchi; A.C.D.Bregeon; D.Moras; A.Takenaka
通讯作者：
A.Takenaka

Insights into the DNA stabilizing contributions of a bicyclic cytosine analogue: crystal structures of DNA duplex containing 7,8-dihydropyrido[2,3-d]pyrimidin-2-on

深入了解双环胞嘧啶类似物的 DNA 稳定作用：含有 7,8-二氢吡啶并[2,3-d]嘧啶-2-on 的 DNA 双链体的晶体结构

DOI：
发表时间：
2010
期刊：
影响因子：
0
作者：
Ella CzarinaMagat Juan; Satoru Shimizu; Xiao Ma; Taizo Kurose; Tsuyoshi Haraguchi1; Fang Zhang; Masaru Tsunoda; Akihiro Ohkubo; Mitsuo Sekine; Takayuki Shibata; Christopher L. Millington; David M. Williams; Akio Takenaka
通讯作者：
Akio Takenaka

Crystallo-graphic study of wild type carbonic anhydrase αCA1 from Chlamydomonas reinhardtii

莱茵衣藻野生型碳酸酐酶αCA1的晶体学研究

DOI：
发表时间：
2010
期刊：
影响因子：
0
作者：
Kaoru Suzuki; Satoru Shimizu; Ella Czarina Magat Juan; Takahiro Miyamoto; Fang Zhang; Md. Mominul Hoque; Yoshiteru Sato; Masaru Tsunoda; Takeshi Sekiguchi; Akio Takenaka
通讯作者：
Akio Takenaka

Insights into the DNA stabilizing contributions of a bicyclic cytosine analogue : crystal structures of DNA duplex containing 7,8-dihydropyrido[2,3-d]pyrimidin-2-on

深入了解双环胞嘧啶类似物的 DNA 稳定作用：含有 7,8-二氢吡啶并[2,3-d]嘧啶-2-on 的 DNA 双链体的晶体结构