Department of Biology, Faculty of Science, Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata 950-2181, Japan.
Department of Biology, Faculty of Science, Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata 950-2181, Japan.
J Mol Biol. 2021 Jul 23;433(15):167046. doi: 10.1016/j.jmb.2021.167046. Epub 2021 May 8.
The ribosomal stalk protein plays a crucial role in functional interactions with translational GTPase factors. It has been shown that the archaeal stalk aP1 binds to both GDP- and GTP-bound conformations of aEF1A through its C-terminal region in two different modes. To obtain an insight into how the aP1•aEF1A binding mode changes during the process of nucleotide exchange from GDP to GTP on aEF1A, we have analyzed structural changes in aEF1A upon binding of the nucleotide exchange factor aEF1B. The isolated archaeal aEF1B has nucleotide exchange ability in the presence of aa-tRNA but not deacylated tRNA, and increases activity of polyphenylalanine synthesis 4-fold. The aEF1B mutation, R90A, results in loss of its original nucleotide exchange activity but retains a remarkable ability to enhance polyphenylalanine synthesis. These results suggest an additional functional role for aEF1B other than in nucleotide exchange. The crystal structure of the aEF1A•aEF1B complex, resolved at 2.0 Å resolution, shows marked rotational movement of domain 1 of aEF1A compared to the structure of aEF1A•GDP•aP1, and this conformational change results in disruption of the original aP1 binding site between domains 1 and 3 of aEF1A. The loss of aP1 binding to the aEF1A•aEF1B complex was confirmed by native gel analysis. The results suggest that aEF1B plays a role in switching off the interaction between aP1 and aEF1A•GDP, as well as in nucleotide exchange, and promote translation elongation.
核糖体柄蛋白在与翻译 GTP 酶因子的功能相互作用中起着至关重要的作用。已经表明，古菌柄蛋白 aP1 通过其 C 末端区域以两种不同的模式结合到 aEF1A 的 GDP 和 GTP 结合构象。为了深入了解 aP1•aEF1A 结合模式在 aEF1A 上 GDP 到 GTP 的核苷酸交换过程中如何变化，我们分析了 aEF1A 与核苷酸交换因子 aEF1B 结合时结构的变化。分离的古菌 aEF1B 在存在 aa-tRNA 但不存在去酰化 tRNA 的情况下具有核苷酸交换能力，并将多苯丙氨酸合成的活性提高了 4 倍。aEF1B 的 R90A 突变导致其原始核苷酸交换活性丧失，但仍保留显著增强多苯丙氨酸合成的能力。这些结果表明 aEF1B 除了在核苷酸交换中具有额外的功能作用。分辨率为 2.0 Å 的 aEF1A•aEF1B 复合物的晶体结构显示，与 aEF1A•GDP•aP1 的结构相比，aEF1A 的结构域 1 发生了明显的旋转运动，这种构象变化导致 aEF1A 的结构域 1 和 3 之间的原始 aP1 结合位点被破坏。通过 native gel 分析证实了 aP1 与 aEF1A•aEF1B 复合物的结合丢失。结果表明，aEF1B 在关闭 aP1 与 aEF1A•GDP 的相互作用以及核苷酸交换中发挥作用，并促进翻译延伸。
