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

• 成果类型: Article
• 署名作者: Baldwin, Eric T.; van Eeuwen, Trevor; Hoyos, David; Zalevsky, Arthur; Tchesnokov, Egor P.; Sanchez, Roberto; Miller, Bryant D.; Di Stefano, Luciano H.; Ruiz, Francesc Xavier; Hancock, Matthew; Isik, 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.; Goette, Matthias; Rout, Michael P.; Arnold, Eddy; Greenbaum, Benjamin D.; Romero, Donna L.; Lacava, John; Taylor, Martin S.
• 署名单位: Rockefeller University; Memorial Sloan Kettering Cancer Center; University of California System; University of California San Francisco; University of California System; University of California San Francisco; University of California System; University of California San Francisco; University of Alberta; Harvard University; Harvard University Medical Affiliates; Dana-Farber Cancer Institute; Harvard University; Harvard Medical School; University of Groningen; Rutgers University System; Rutgers University New Brunswick; Rutgers University System; Rutgers University New Brunswick; Massachusetts Institute of Technology (MIT); Whitehead Institute; Stanford University; Stanford University; Stanford Cancer Institute; Stanford University; Max Planck Society; Johns Hopkins University; University of Texas System; University of Texas Arlington; Cornell University; Weill Cornell Medicine; Harvard University; Harvard University Medical Affiliates; Massachusetts General Hospital
• 刊物名称: Nature
• ISSN/ISSBN: 0028-3862
• DOI: 10.1038/s41586-023-06947-z
• 发表日期: 2024-02-01
• 关键词: transcriptase inhibitor nnrti; hiv-1 reverse-transcriptase; human l1 retrotransposition; crystal-structure; dna; rna; resistance; endonuclease; identification; mechanisms

摘要

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 catalysed 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 ageing6,7, 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-electron microscopy in several conformational states. Our analyses identified two previously undescribed 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 show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates 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 the evolutionary history of L1 and enable rational drug development targeting L1. X-ray crystallography, cryo-electron microscopy, structural modelling, biochemistry, cell biology, and evolutionary analysis enable characterization of ORF2p, the reverse transcriptase of the ancient 'parasitic' LINE-1 retrotransposon that has written around one-third of the human genome.