Crystal structures of the CP1 domain from Thermus thermophilus isoleucyl-tRNA synthetase and its complex with L-valine
Crystal structures of the CP1 domain from Thermus thermophilus isoleucyl-tRNA synthetase and its complex with L-valine
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DOI：
10.1074/jbc.m312830200
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发表时间：
2004-02-27
影响因子：
4.8
通讯作者：
Yokoyama, S
中科院分区：
文献类型：
--
作者：
Fukunaga, R;Fukai, S;Yokoyama, S
异亮氨酰-tRNA 合成酶 (IleRS) 将 tRNA(Ile) 不仅与其同源异亮氨酸连接起来，而且还与近同源的缬氨酸连接。 IleRS 的 CP1 结构域可脱酰化或编辑错误充电的 Val-tRNA(Ile)。我们分别以 1.8 埃和 2.0 埃的分辨率确定了单独的嗜热栖热菌 IleRS CP1 结构域及其与缬氨酸复合物的晶体结构。在复杂的结构中，Asp(328) 残基可识别缬氨酸 NH3+ 基团，先前的突变分析表明，Asp(328) 残基对于针对 Val-tRNA(Ile) 的编辑反应至关重要。缬氨酸侧链结合口袋仅足够大以容纳缬氨酸，并且在该位置放置异亮氨酸模型表明异亮氨酸的额外亚甲基会与His(319)发生冲突。据报道，大肠杆菌 IleRS 的 H319A 突变体除了 Val-tRNA(Ile) 之外，还使同源 Ile-tRNA(Ile) 脱酰，表明本研究中发现的缬氨酸结合模式代表了 Val-tRNA(Ile) 编辑反应中的模式。对嗜热嗜热菌 IleRS 突变体的 Val-tRNA(Ile) 编辑活性的分析揭示了 Thr(228)、Thr(229)、Thr(230) 和 Asp(328) 的重要性，它们与当前结构中的水分子协调。 IleRS 编辑位点结合的 Val-tRNA(Ile) 的 Val-腺苷部分的结构模型揭示了 IleRS 和嗜热链霉亮氨酰-tRNA 合成酶之间底物结合和识别机制的一些有趣差异。例如，氨基酸的羰基氧相对于腺苷核糖环彼此相对，并且被不同地识别。
Isoleucyl-tRNA synthetase (IleRS) links tRNA(Ile) with not only its cognate isoleucine but also the nearly cognate valine. The CP1 domain of IleRS deacylates, or edits, the mischarged Val-tRNA(Ile). We determined the crystal structures of the Thermus thermophilus IleRS CP1 domain alone, and in its complex with valine at 1.8- and 2.0-Angstrom resolutions, respectively. In the complex structure, the Asp(328) residue, which was shown to be critical for the editing reaction against Val-tRNA(Ile) by a previous mutational analysis, recognizes the valine NH3+ group. The valine side chain binding pocket is only large enough to accommodate valine, and the placement of an isoleucine model in this location revealed that the additional methylene group of isoleucine would clash with His(319). The H319A mutant of Escherichia coli IleRS reportedly deacylates the cognate Ile-tRNA(Ile) in addition to Val-tRNA(Ile), indicating that the valine-binding mode found in this study represents that in the Val-tRNA(Ile) editing reaction. Analyses of the Val-tRNA(Ile) editing activities of T. thermophilus IleRS mutants revealed the importance of Thr(228), Thr(229), Thr(230), and Asp(328), which are coordinated with water molecules in the present structure. The structural model for the Val-adenosine moiety of Val-tRNA(Ile) bound in the IleRS editing site revealed some interesting differences in the substrate binding and recognizing mechanisms between IleRS and T. thermophilus leucyl-tRNA synthetase. For example, the carbonyl oxygens of the amino acids are located opposite to each other, relative to the adenosine ribose ring, and are differently recognized.