Nucleic-acid-triggered NADase activation of a short prokaryotic Argonaute

Xiaopan Gao, Kun Shang, Kaixiang Zhu, Linyue Wang, Zhixia Mu, Xingke Fu, Xia Yu, Bo Qin, Hongtao Zhu (), Wei Ding () and Sheng Cui ()
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Xiaopan Gao: Chinese Academy of Medical Sciences and Peking Union Medical College
Kun Shang: Chinese Academy of Sciences
Kaixiang Zhu: Chinese Academy of Medical Sciences and Peking Union Medical College
Linyue Wang: Chinese Academy of Medical Sciences and Peking Union Medical College
Zhixia Mu: Chinese Academy of Medical Sciences and Peking Union Medical College
Xingke Fu: Chinese Academy of Sciences
Xia Yu: Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute
Bo Qin: Chinese Academy of Medical Sciences and Peking Union Medical College
Hongtao Zhu: Chinese Academy of Sciences
Wei Ding: Chinese Academy of Sciences
Sheng Cui: Chinese Academy of Medical Sciences and Peking Union Medical College

Nature, 2024, vol. 625, issue 7996, 822-831

Abstract: Abstract Argonaute (Ago) proteins mediate RNA- or DNA-guided inhibition of nucleic acids1,2. Although the mechanisms used by eukaryotic Ago proteins and long prokaryotic Ago proteins (pAgos) are known, that used by short pAgos remains elusive. Here we determined the cryo-electron microscopy structures of a short pAgo and the associated TIR-APAZ proteins (SPARTA) from Crenotalea thermophila (Crt): a free-state Crt-SPARTA; a guide RNA–target DNA-loaded Crt-SPARTA; two Crt-SPARTA dimers with distinct TIR organization; and a Crt-SPARTA tetramer. These structures reveal that Crt-SPARTA is composed of a bilobal-fold Ago lobe that connects with a TIR lobe. Whereas the Crt-Ago contains a MID and a PIWI domain, Crt-TIR-APAZ has a TIR domain, an N-like domain, a linker domain and a trigger domain. The bound RNA–DNA duplex adopts a B-form conformation that is recognized by base-specific contacts. Nucleic acid binding causes conformational changes because the trigger domain acts as a ‘roadblock’ that prevents the guide RNA 5′ ends and the target DNA 3′ ends from reaching their canonical pockets; this disorders the MID domain and promotes Crt-SPARTA dimerization. Two RNA–DNA-loaded Crt-SPARTA dimers form a tetramer through their TIR domains. Four Crt-TIR domains assemble into two parallel head-to-tail-organized TIR dimers, indicating an NADase-active conformation, which is supported by our mutagenesis study. Our results reveal the structural basis of short-pAgo-mediated defence against invading nucleic acids, and provide insights for optimizing the detection of SPARTA-based programmable DNA sequences.

Date: 2024
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DOI: 10.1038/s41586-023-06665-6

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