Identification of borrelidin binding site on threonyl-tRNA synthetase.
Identification of borrelidin binding site on threonyl-tRNA synthetase.
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DOI：
10.1016/j.bbrc.2014.07.100
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发表时间：
2014-09
影响因子：
3.1
通讯作者：
Ming Li;Ji Zhang;Chongxi Liu;Baozhu Fang;Xiangjing Wang;W. Xiang
中科院分区：
文献类型：
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作者：
Ming Li;Ji Zhang;Chongxi Liu;Baozhu Fang;Xiangjing Wang;W. Xiang
冬凌草素具有广泛的生物活性，被认为是苏氨酰-tRNA合成酶(ThrRS)的非竞争性抑制剂。然而，目前关于Borreldin抗ThrRS的详细机制，尤其是其与ThrRs结合部位的研究尚不清楚，这限制了新型Borrelin衍生物的开发和合理设计构型ThrRS抑制剂。在本研究中，通过分子对接的方法预测了冬凌草素与大肠杆菌ThrRS的结合部位。为了验证我们的推测，E。构建了P424K、E458Δ和G459Δ，并通过酶动力学和停流荧光分析比较了它们与野生型ThrRS的敏感性。对接结果表明，冬凌草素结合在ThrRs活性部位外面但邻近的口袋，由Y313、R363、R375、P424、E458、G459和K465残基组成。定点突变结果表明，P424K、E458Δ和G459ΔThrRS对冬凌草素的敏感性显著降低。所有结果表明，Y313、P424、E458和G459残基在B蛋白与ThrRS结合过程中起重要作用。这表明，冬凌草素可能通过抑制ThrRs的活性而不是抑制ATP与苏氨酸的结合，而诱导裂隙闭合，从而阻断Thr-AMP和PPI的释放。这一研究为进一步研究冬凌草素对ThrRs的抑制机制提供了新的思路。
Borrelidin exhibits a wide spectrum of biological activities and has been considered as a non-competitive inhibitor of threonyl-tRNA synthetase (ThrRS). However, the detailed mechanisms of borrelidin against ThrRS, especially borrelidin binding site on ThrRS, are still unclear, which limits the development of novel borrelidin derivatives and rational design of structure-based ThrRS inhibitors. In this study, the binding site of borrelidin onEscherichia coliThrRS was predicted by molecular docking. To validate our speculations, the ThrRS mutants ofE. coli(P424K, E458Δ, and G459Δ) were constructed and their sensitivity to borrelidin was compared to that of the wild-type ThrRS by enzyme kinetics and stopped-flow fluorescence analysis. The docking results showed that borrelidin binds the pocket outside but adjacent to the active site of ThrRS, consisting of residue Y313, R363, R375, P424, E458, G459, and K465. Site-directed mutagenesis results showed that sensitivities of P424K, E458Δ, and G459Δ ThrRSs to borrelidin were reduced markedly. All the results showed that residue Y313, P424, E458, and G459 play vital roles in the binding of borrelidin to ThrRS. It indicated that borrelidin may induce the cleft closure, which blocks the release of Thr-AMP and PPi, to inhibit activity of ThrRS rather than inhibit the binding of ATP and threonine. This study provides new insight into inhibitory mechanisms of borrelidin against ThrRS.