Department of Biological Sciences, Marquette University, Milwaukee, WI 53233, USA.
Department of Immunology, The University of Iowa, Iowa City, IA 52242, USA.
Nucleic Acids Res. 2023 Feb 28;51(4):1803-1822. doi: 10.1093/nar/gkac1266.
Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.
核糖体亚基的组装形成活性核糖体复合物是蛋白质合成的关键步骤。eIF6 从 60S 核糖体亚基上的释放为 60S 与 40S 亚基的结合和参与翻译做好了准备。eIF6 与 60S 的 uL14（RPL23）界面的相互作用动力学及其受 Shwachman-Diamond 综合征（SDS）中获得的体细胞突变的干扰尚未得到明确理解。在这里，通过使用一种改良的策略获得高产量的重组人 eIF6，我们揭示了关键界面，该界面涉及 uL14 的 C 尾中的八个关键残基，对于 60S 和 eIF6 之间的物理相互作用至关重要。eIF6 疾病变异体的构象变化破坏了互补结合界面，为变异体与 60S 之间的相互作用减弱提供了一种机制。氢氘交换质谱（HDX-MS）分析揭示了 eIF6 与 uL14 结合诱导的 eIF6 中的动态构象重排，并暴露了由 eIF6 的 C 尾调节的变构界面。破坏 eIF6-60S 结合界面中的关键残基显著限制了癌细胞的增殖，这突出了针对该界面进行治疗的重要性。因此，确定这些关键界面为在癌症和 SDS 中靶向 eIF6 提供了治疗框架。
