Structures of a putative RNA 5-methyluridine methyltransferase, Thermus thermophilus TTHA1280, and its complex with S-adenosyl-L-homocysteine.
Structures of a putative RNA 5-methyluridine methyltransferase, Thermus thermophilus TTHA1280, and its complex with S-adenosyl-L-homocysteine.
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DOI：
10.1107/s1744309105029842
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发表时间：
2005-10
期刊：
影响因子：
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通讯作者：
A. Pioszak;K. Murayama;N. Nakagawa;A. Ebihara;S. Kuramitsu;M. Shirouzu;S. Yokoyama
中科院分区：
文献类型：
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作者：
A. Pioszak;K. Murayama;N. Nakagawa;A. Ebihara;S. Kuramitsu;M. Shirouzu;S. Yokoyama
嗜热栖热菌假设蛋白 TTHA1280 属于预测的 S-腺苷-L-甲硫氨酸 (AdoMet) 依赖性 RNA 甲基转移酶 (MTase) 家族，存在于许多细菌和古细菌物种中。对 I 类罗斯曼折叠样 MTase 共有的氨基酸序列基序的检查表明其作为 RNA 5-甲基尿苷 MTase 具有特定作用。表达、纯化和结晶硒代蛋氨酸 (SeMet) 标记的蛋白质和天然版本的蛋白质。获得了 SeMet 标记的脱辅基蛋白的两种晶型：SeMet-ApoI 和 SeMet-ApoII。天然蛋白与 S-腺苷-L-高半胱氨酸 (AdoHcy) 共结晶产生了第三种晶体形式，Native-AdoHcy。 SeMet-ApoI 结构通过多重反常色散法解析，并以 2.55 A 分辨率进行精修。 SeMet-ApoII 和 Native-AdoHcy 结构通过分子置换解析，并分别在 1.80 和 2.60 A 下精炼。 TTHA1280 在晶体和溶液中形成同二聚体。每个亚基折叠成一个三结构域结构，由一个小的 N 端 PUA 结构域、一个中央 α/β 结构域和一个 C 端罗斯曼折叠样 MTase 结构域组成。这三个结构域形成了一个整体钳状形状，假定的活性位点面向深裂口。活性位点的结构与尿苷的特异性识别和甲基转移至5碳位的催化作用一致。裂口的大小和电荷分布适合结合单链RNA。
The Thermus thermophilus hypothetical protein TTHA1280 belongs to a family of predicted S-adenosyl-L-methionine (AdoMet) dependent RNA methyltransferases (MTases) present in many bacterial and archaeal species. Inspection of amino-acid sequence motifs common to class I Rossmann-fold-like MTases suggested a specific role as an RNA 5-methyluridine MTase. Selenomethionine (SeMet) labelled and native versions of the protein were expressed, purified and crystallized. Two crystal forms of the SeMet-labelled apoprotein were obtained: SeMet-ApoI and SeMet-ApoII. Cocrystallization of the native protein with S-adenosyl-L-homocysteine (AdoHcy) yielded a third crystal form, Native-AdoHcy. The SeMet-ApoI structure was solved by the multiple anomalous dispersion method and refined at 2.55 A resolution. The SeMet-ApoII and Native-AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60 A, respectively. TTHA1280 formed a homodimer in the crystals and in solution. Each subunit folds into a three-domain structure composed of a small N-terminal PUA domain, a central alpha/beta-domain and a C-terminal Rossmann-fold-like MTase domain. The three domains form an overall clamp-like shape, with the putative active site facing a deep cleft. The architecture of the active site is consistent with specific recognition of uridine and catalysis of methyl transfer to the 5-carbon position. The cleft is suitable in size and charge distribution for binding single-stranded RNA.