期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：31
DOI：10.1073/pnas.2201662119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Shelterin is a multiprotein complex that plays central roles in telomere biology. Mutations in shelterin result in premature aging diseases and familial cancer predisposition. Mechanistic understanding of these so-called telomereopathies is hampered by our lack of knowledge regarding the structure and stoichiometry of shelterin. Here, we use multiple methods to probe the stoichiometry and conformational states of shelterin and reveal that it forms a fully dimeric complex with extensive conformational heterogeneity. Our results highlight the dynamic nature of this essential complex and explain why its high-resolution structure determination has yet to be achieved.
Human shelterin is a six-subunit complex—composed of TRF1, TRF2, Rap1, TIN2, TPP1, and POT1—that binds telomeres, protects them from the DNA-damage response, and regulates the maintenance of telomeric DNA. Although high-resolution structures have been generated of the individual structured domains within shelterin, the architecture and stoichiometry of the full complex are currently unknown. Here, we report the purification of shelterin subcomplexes and reconstitution of the entire complex using full-length, recombinant subunits. By combining negative-stain electron microscopy (EM), cross-linking mass spectrometry (XLMS), AlphaFold modeling, mass photometry, and native mass spectrometry (MS), we obtain stoichiometries as well as domain-scale architectures of shelterin subcomplexes and determine that they feature extensive conformational heterogeneity. For POT1/TPP1 and POT1/TPP1/TIN2, we observe high variability in the positioning of the POT1 DNA-binding domain, the TPP1 oligonucleotide/oligosaccharide–binding (OB) fold, and the TIN2 TRFH domain with respect to the C-terminal domains of POT1. Truncation of unstructured linker regions in TIN2, TPP1, and POT1 did not reduce the conformational variability of the heterotrimer. Shelterin and TRF1-containing subcomplexes form fully dimeric stoichiometries, even in the absence of DNA substrates. Shelterin and its subcomplexes showed extensive conformational variability, regardless of the presence of DNA substrates. We conclude that shelterin adopts a multitude of conformations and argue that its unusual architectural variability is beneficial for its many functions at telomeres.
