Department of Integrated Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire, INSERM U964, CNRS UMR7104, Université de Strasbourg, 67404 Illkirch, France.
Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY 10065.
Proc Natl Acad Sci U S A. 2017 Dec 19;114(51):E10899-E10908. doi: 10.1073/pnas.1715501114. Epub 2017 Dec 5.
Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages (initiation, elongation, termination, and recycling) of the translation mechanism. The propensity of aminoglycosides to induce miscoding errors that suppress the termination of protein synthesis supports their potential as therapeutic interventions in human diseases associated with premature termination codons (PTCs). However, the sites of interaction of aminoglycosides with the eukaryotic ribosome and their modes of action in eukaryotic translation remain largely unexplored. Here, we use the combination of X-ray crystallography and single-molecule FRET analysis to reveal the interactions of distinct classes of aminoglycosides with the 80S eukaryotic ribosome. Crystal structures of the 80S ribosome in complex with paromomycin, geneticin (G418), gentamicin, and TC007, solved at 3.3- to 3.7-Å resolution, reveal multiple aminoglycoside-binding sites within the large and small subunits, wherein the 6'-hydroxyl substituent in ring I serves as a key determinant of binding to the canonical eukaryotic ribosomal decoding center. Multivalent binding interactions with the human ribosome are also evidenced through their capacity to affect large-scale conformational dynamics within the pretranslocation complex that contribute to multiple aspects of the translation mechanism. The distinct impacts of the aminoglycosides examined suggest that their chemical composition and distinct modes of interaction with the ribosome influence PTC read-through efficiency. These findings provide structural and functional insights into aminoglycoside-induced impacts on the eukaryotic ribosome and implicate pleiotropic mechanisms of action beyond decoding.
氨基糖苷类是化学性质多样的广谱抗生素，作用于细菌核糖体的功能中心，从而影响翻译机制的所有四个主要阶段（起始、延伸、终止和循环）。氨基糖苷类具有诱导错义错误的倾向，从而抑制蛋白质合成的终止，这支持了它们在与过早终止密码子（PTC）相关的人类疾病中作为治疗干预的潜力。然而，氨基糖苷类与真核核糖体的相互作用位点及其在真核翻译中的作用模式在很大程度上仍未得到探索。在这里，我们使用 X 射线晶体学和单分子 FRET 分析相结合的方法，揭示了不同类别的氨基糖苷类与 80S 真核核糖体的相互作用。通过解决 3.3-至 3.7-Å 分辨率的帕罗霉素、遗传霉素（G418）、庆大霉素和 TC007 与 80S 核糖体复合物的晶体结构，揭示了大亚基和小亚基内存在多个氨基糖苷类结合位点，其中 I 环上的 6'-羟基取代基是与经典真核核糖体解码中心结合的关键决定因素。通过它们影响前移位复合物内的大尺度构象动力学的能力，与人类核糖体的多价结合相互作用也得到了证明，这有助于翻译机制的多个方面。所检查的氨基糖苷类的不同影响表明，它们的化学组成和与核糖体的不同相互作用模式影响 PTC 通读效率。这些发现为氨基糖苷类对真核核糖体的影响提供了结构和功能见解，并暗示了除解码之外的多种作用机制。
