近日，美国哈佛医学院Martin S. Taylor等研究人员合作揭示人类LINE-1 ORF2蛋白的结构、功能和适应性。2023年12月14日，国际知名学术期刊《自然》在线发表了这一成果。

研究人员表示，LINE-1（L1）反转录转座子是一种古老的基因寄生虫，通过其多功能酶——开放阅读框2蛋白（ORF2p）来催化的“复制-粘贴”机制，写入了约三分之一的人类基因组。ORF2p的反转录酶（RT）和内切酶活性与癌症、自身免疫和衰老的病理生理学有关，因此ORF2p成为潜在的治疗靶点。然而，结构和机理知识的缺乏阻碍了对其进行合理利用的努力。

研究人员通过X射线晶体学和冷冻电镜，报告了人类ORF2p“核心”（残基238-1061位，包括RT结构域）在多种构象状态下的结构。这些分析揭示了两个新的折叠结构域、与RNA模板的广泛接触以及有助于L1复制周期独特方面的相关适应性。全长ORF2p的计算整合结构模型显示了一种动态闭环构象，这种构象似乎在逆转录过程中打开。研究人员描述了ORF2p RT抑制的特征，并揭示了其潜在的结构基础。成像和生物化学发现，非经典的胞浆ORF2p RT活性可产生RNA:DNA杂交体，通过cGAS/STING激活先天性免疫信号，导致干扰素产生。与逆转录病毒RT不同的是，L1 RT能有效地由短RNA和发夹引物引出，这可能是胞质活化的原因。

其他生化活动包括加工性、DNA定向聚合、非模板化碱基添加和模板切换，这些使研究人员能够提出一个更新的L1插入模型。最后，演化分析揭示了ORF2p与其他RNA依赖性聚合酶和DNA依赖性聚合酶之间的结构保守性。因此，研究人员为L1聚合和插入提供了关键的机理见解，揭示了L1的演化历史，并促进了针对L1的合理药物开发。

附：英文原文

Title: Structures, functions, and adaptations of the human LINE-1 ORF2 protein

Author: Baldwin, Eric T., van Eeuwen, Trevor, Hoyos, David, Zalevsky, Arthur, Tchesnokov, Egor P., Snchez, Roberto, Miller, Bryant D., Di Stefano, Luciano H., Ruiz, Francesc Xavier, Hancock, Matthew, Iik, Esin, Mendez-Dorantes, Carlos, Walpole, Thomas, Nichols, Charles, Wan, Paul, Riento, Kirsi, Halls-Kass, Rowan, Augustin, Martin, Lammens, Alfred, Jestel, Anja, Upla, Paula, Xibinaku, Kera, Congreve, Samantha, Hennink, Maximiliaan, Rogala, Kacper B., Schneider, Anna M., Fairman, Jennifer E., Christensen, Shawn M., Desrosiers, Brian, Bisacchi, Gregory S., Saunders, Oliver L., Hafeez, Nafeeza, Miao, Wenyan, Kapeller, Rosana, Zaller, Dennis M., Sali, Andrej, Weichenrieder, Oliver, Burns, Kathleen H., Gtte, Matthias, Rout, Michael P., Arnold, Eddy, Greenbaum, Benjamin D., Romero, Donna L., LaCava, John, Taylor, Martin S.

Issue&Volume: 2023-12-14

Abstract: The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one third of the human genome through a “copy-and-paste” mechanism catalyzed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5, and aging6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p ‘core’ (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-EM in multiple conformational states. Our analyses reveal two novel folded domains, extensive contacts to RNA templates, and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry reveal that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signaling via cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which likely explains cytosolic priming. Additional biochemical activities including processivity, DNA-directed polymerization, non-templated base addition, and template switching together allow us to propose an updated L1 insertion model. Finally, our evolutionary analysis reveals structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on L1 evolutionary history, and enable rational drug development targeting L1.

DOI: 10.1038/s41586-023-06947-z

Source: https://www.nature.com/articles/s41586-023-06947-z