Graduate School of Science, Osaka City University, Japan.
Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.
FEBS Lett. 2021 Jan;595(2):264-274. doi: 10.1002/1873-3468.13996. Epub 2020 Nov 28.
Thermophilic proteins maintain their structure at high temperatures through a combination of various factors. Here, we report the ligand-induced stabilization of a thermophilic Ser/Thr protein kinase. Thermus thermophilus TpkD unfolds completely at 55 °C despite the optimum growth temperature of 75 °C. Unexpectedly, we found that the TpkD structure is drastically stabilized by its natural ligands ATP and ADP, as evidenced by the increase in the melting temperature to 80 °C. Such a striking effect of a substrate on thermostability has not been reported for other protein kinases. Conformational changes upon ATP binding were observed in fluorescence quenching and limited proteolysis experiments. Urea denaturation of Trp mutants suggested that ATP binding affects not only the ATP-binding site, but also the remote regions. Our findings shed light on thermoadaptation of thermophilic proteins.
嗜热蛋白通过各种因素的结合来维持其在高温下的结构。在这里，我们报告了一种嗜热丝氨酸/苏氨酸蛋白激酶在配体诱导下的稳定化。尽管最适生长温度为 75°C，但嗜热栖热菌 TpkD 在 55°C 时完全展开。出乎意料的是，我们发现 TpkD 结构通过其天然配体 ATP 和 ADP 得到了极大的稳定，这表现在其熔点增加到 80°C。这种底物对热稳定性的显著影响在其他蛋白激酶中尚未报道过。在荧光猝灭和有限蛋白水解实验中观察到了 ATP 结合时的构象变化。色氨酸突变体的脲变性表明，ATP 结合不仅影响 ATP 结合位点，还影响远程区域。我们的发现揭示了嗜热蛋白的热适应机制。
