Role of Coupled Dynamics in the Catalytic Activity of Prokaryotic-like Prolyl-tRNA Synthetases
Role of Coupled Dynamics in the Catalytic Activity of Prokaryotic-like Prolyl-tRNA Synthetases
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DOI：
10.1021/bi300097g
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发表时间：
2012-03-13
期刊：
影响因子：
2.9
通讯作者：
Hati, Sanchita
中科院分区：
文献类型：
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作者：
Sanford, Brianne;Cao, Bach;Hati, Sanchita
脯氨酸-tRNA合成酶（prors）已被证明可以激活同源氨基酸和一些非同源氨基酸，并将它们附着在特定的tRNA（Pro）底物上。例如，丙氨酸比同源脯氨酸小，被大肠杆菌ProRS失活。错载的Ala-tRNA（Pro）被不同于激活结构域的编辑结构域（INS）水解。先前的研究表明，INS的缺失大大降低了同源脯氨酸的激活效率。在本研究中，我们采用实验和计算方法来验证INS的缺失会改变内部蛋白质动力学从而导致催化功能降低的假设。对两个pros变体G217A和E218A的动力学研究显示，氨基酸激活效率降低。分子动力学研究表明，INS与含有具有重要催化作用的脯氨酸结合环（PBL，残基199-206）的蛋白质片段之间存在运动耦合。特别是INS的完全缺失，以及G217或E218对丙氨酸的突变，对PBL的运动有显著影响。通过硅突变和本质动力学分析，还观察到相邻蛋白质片段之间存在耦合动力学。总之，本研究表明，编辑域-激活域界面上的结构元件参与了促进氨基酸结合和细菌prors催化的耦合运动，这可能解释了为什么尽管缺乏催化活性，但在某些系统中仍保持了截断或失效的编辑域。
Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNA(Pro) substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNA(Pro) is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In this study, experimental and computational approaches were used to test the hypothesis that deletion of the INS alters the internal protein dynamics leading to reduced catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199-206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Altogether, this study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity.