Energy landscape of domain motion in glutamate dehydrogenase deduced from cryo‐electron microscopy
Energy landscape of domain motion in glutamate dehydrogenase deduced from cryo‐electron microscopy
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DOI：
10.1111/febs.15224
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发表时间：
2020-01
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影响因子：
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通讯作者：
Mao Oide;Takayuki Kato;Tomotaka Oroguchi;M. Nakasako
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作者：
Mao Oide;Takayuki Kato;Tomotaka Oroguchi;M. Nakasako
蛋白质构象变化的分析对于阐明与其功能相关的蛋白质运动机制非常重要。在这里，我们使用冷冻电子显微镜研究了深热球菌中未配体的谷氨酸脱氢酶的自发结构域运动，并提出了一种构建蛋白质构象自由能景观的新方法。同源六聚体酶的每个亚基均包含核苷酸结合域（NAD 域）和六聚体形成核心域。一个大的活性位点裂缝位于两个域之间，并根据 NAD 域的运动从打开到关闭变化。从所有冷冻电子显微镜图像重建的三维图显示了 NAD 结构域的无序体积，这表明收集到的图像中的 NAD 结构域在结构域运动中采用了不同的构象。对亚基 NAD 域的集中分类提供了域运动中可能构象的几个图。为了推断 EM 图像中出现了哪些构象，我们开发了一种新颖的分析方法，将 EM 图描述为 200 ns 分子动力学模拟中出现的代表性构象的线性组合作为参考。该分析使我们能够估计代表性构象的出现频率，这说明了域运动中的自由能景观。在开闭域运动中，两个自由能盆阻碍了从开态到闭态的直接转变。为分类中的代表性电磁图构建的结构模型证明了能量景观与域运动构象之间的相关性。基于这些结果，讨论了谷氨酸脱氢酶的结构域运动以及可视化构象变化和自由能景观的分析方法。
Analysis of the conformational changes of protein is important to elucidate the mechanisms of protein motions correlating with their function. Here, we studied the spontaneous domain motion of unliganded glutamate dehydrogenase from Thermococcus profundus using cryo‐electron microscopy and proposed a novel method to construct free‐energy landscape of protein conformations. Each subunit of the homo‐hexameric enzyme comprises nucleotide‐binding domain (NAD domain) and hexamer‐forming core domain. A large active‐site cleft is situated between the two domains and varies from open to close according to the motion of a NAD domain. A three‐dimensional map reconstructed from all cryo‐electron microscopy images displayed disordered volumes of NAD domains, suggesting that NAD domains in the collected images adopted various conformations in domain motion. Focused classifications on NAD domain of subunits provided several maps of possible conformations in domain motion. To deduce what kinds of conformations appeared in EM images, we developed a novel analysis method that describe the EM maps as a linear combination of representative conformations appearing in a 200‐ns molecular dynamics simulation as reference. The analysis enabled us to estimate the appearance frequencies of the representative conformations, which illustrated a free‐energy landscape in domain motion. In the open/close domain motion, two free‐energy basins hindered the direct transformation from open to closed state. Structure models constructed for representative EM maps in classifications demonstrated the correlation between the energy landscape and conformations in domain motion. Based on the results, the domain motion in glutamate dehydrogenase and the analysis method to visualize conformational changes and free‐energy landscape were discussed.