Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden.
Department of Cell and Molecular Biology, Uppsala University, 75124 Uppsala, Sweden
Proc Natl Acad Sci U S A. 2018 May 1;115(18):4649-4654. doi: 10.1073/pnas.1802001115. Epub 2018 Apr 23.
The interaction between the ribosomal-stalk protein L7/12 (L12) and initiation factor 2 (IF2) is essential for rapid subunit association, but the underlying mechanism is unknown. Here, we have characterized the L12-IF2 interaction on ribosomes using site-directed mutagenesis, fast kinetics, and molecular dynamics (MD) simulations. Fifteen individual point mutations were introduced into the C-terminal domain of L12 (L12-CTD) at helices 4 and 5, which constitute the common interaction site for translational GTPases. In parallel, 15 point mutations were also introduced into IF2 between the G4 and G5 motifs, which we hypothesized as the potential L12 interaction sites. The L12 and IF2 mutants were tested in ribosomal subunit association assay in a stopped-flow instrument. Those amino acids that caused defective subunit association upon substitution were identified as the molecular determinants of L12-IF2 interaction. Further, MD simulations of IF2 docked onto the L12-CTD pinpointed the exact interacting partners-all of which were positively charged on L12 and negatively charged on IF2, connected by salt bridges. Lastly, we tested two pairs of charge-reversed mutants of L12 and IF2, which significantly restored the yield and the rate of formation of the 70S initiation complex. We conclude that complementary charge-based interaction between L12-CTD and IF2 is the key for fast subunit association. Considering the homology of the G domain, similar mechanisms may apply for L12 interactions with other translational GTPases.
核糖体柄蛋白 L7/12（L12）与起始因子 2（IF2）之间的相互作用对于快速亚基缔合至关重要，但潜在的机制尚不清楚。在这里，我们使用定点突变、快速动力学和分子动力学（MD）模拟研究了核糖体上 L12-IF2 相互作用。在构成翻译 GTP 酶常见相互作用位点的 L12（L12-CTD）的螺旋 4 和 5 中，引入了 15 个单独的点突变。同时，在 IF2 的 G4 和 G5 基序之间也引入了 15 个点突变，我们假设这是潜在的 L12 相互作用位点。将 L12 和 IF2 突变体在停流仪器中的核糖体亚基缔合测定中进行了测试。那些取代后导致亚基缔合缺陷的氨基酸被鉴定为 L12-IF2 相互作用的分子决定因素。此外，IF2 对接在 L12-CTD 上的 MD 模拟精确定位了确切的相互作用伙伴-所有这些伙伴在 L12 上带正电荷，在 IF2 上带负电荷，通过盐桥连接。最后，我们测试了 L12 和 IF2 的两对电荷反转突变体，它们显著恢复了 70S 起始复合物的产率和形成速率。我们得出结论，L12-CTD 和 IF2 之间互补的基于电荷的相互作用是快速亚基缔合的关键。考虑到 G 结构域的同源性，类似的机制可能适用于 L12 与其他翻译 GTP 酶的相互作用。
