Interdomain dynamics and coactivation of the mRNA decapping enzyme Dcp2 are mediated by a gatekeeper tryptophan
Interdomain dynamics and coactivation of the mRNA decapping enzyme Dcp2 are mediated by a gatekeeper tryptophan
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DOI：
10.1073/pnas.1113620109
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发表时间：
2012-02-21
影响因子：
11.1
通讯作者：
Gross, John D.
中科院分区：
文献类型：
--
作者：
Floor, Stephen N.;Borja, Mark S.;Gross, John D.
双叶酶的构象动力学可以用来调节它们的活性。一种这样的酶，真核生物脱帽酶Dcp2，通过切割5'帽结构来控制mRNA的半衰期，这暴露了被核酸外切酶有效降解的单磷酸。通过Dcp2的开盖被认为是由开放到封闭的转变控制的，该转变涉及形成具有两个域的复合活性位点，该复合活性位点包围基底，但是该过程的许多细节尚不清楚。在这里，使用NMR光谱和酶动力学，我们表明，Trp43裂殖酵母Dcp2是一个保守的看门人，这个开放到关闭的过渡。我们发现，Dcp2的样品在毫秒微秒的时间尺度上的溶液中的多种构象。突变的看门人色氨酸废除了动态行为的Dcp2和减弱共激活的酵母增强剂的脱帽（Edc1）。我们的研究结果确定了Dcp2的开放到封闭的过渡的动力学，提出了共激活的结构途径，预测Dcp1直接接触Dcp2的催化结构域，并表明Dcp2的脱帽共激活与复合活性位点的形成有关。
Conformational dynamics in bilobed enzymes can be used to regulate their activity. One such enzyme, the eukaryotic decapping enzyme Dcp2, controls the half-life of mRNA by cleaving the 5' cap structure, which exposes a monophosphate that is efficiently degraded by exonucleases. Decapping by Dcp2 is thought to be controlled by an open-to-closed transition involving formation of a composite active site with two domains sandwiching substrate, but many details of this process are not understood. Here, using NMR spectroscopy and enzyme kinetics, we show that Trp43 of Schizosaccharomyces pombe Dcp2 is a conserved gatekeeper of this open-to-closed transition. We find that Dcp2 samples multiple conformations in solution on the millisecond-microsecond timescale. Mutation of the gatekeeper tryptophan abolishes the dynamic behavior of Dcp2 and attenuates coactivation by a yeast enhancer of decapping (Edc1). Our results determine the dynamics of the open-to-closed transition in Dcp2, suggest a structural pathway for coactivation, predict that Dcp1 directly contacts the catalytic domain of Dcp2, and show that coactivation of decapping by Dcp2 is linked to formation of the composite active site.