期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 112, 期 30, 页码 9436-9441出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1508686112
关键词
CoV; nsp14; proofreading; exoribonuclease; methyltransferase
资金
- Ministry of Science and Technology of China Project 973 [2014CB542800, 2014CBA02003]
- National Natural Science Foundation of China [81330036]
- Chinese Academy of Sciences [XDB08020200]
Nonstructural protein 14 (nsp14) of coronaviruses (CoV) is important for viral replication and transcription. The N-terminal exoribonuclease (ExoN) domain plays a proofreading role for prevention of lethal mutagenesis, and the C-terminal domain functions as a (guanine-N7) methyl transferase (N7-MTase) for mRNA capping. The molecular basis of both these functions is unknown. Here, we describe crystal structures of severe acute respiratory syndrome (SARS)-CoV nsp14 in complex with its activator nonstructural protein10 (nsp10) and functional ligands. One molecule of nsp10 interacts with ExoN of nsp14 to stabilize it and stimulate its activity. Although the catalytic core of nsp14 ExoN is reminiscent of proofreading exonucleases, the presence of two zinc fingers sets it apart from homologs. Mutagenesis studies indicate that both these zinc fingers are essential for the function of nsp14. We show that a DEEDh (the five catalytic amino acids) motif drives nucleotide excision. The N7-MTase domain exhibits a noncanonical MTase fold with a rare beta-sheet insertion and a peripheral zinc finger. The cap-precursor guanosine-P3-adenosine-5', 5'-triphosphate and S-adenosyl methionine bind in proximity in a highly constricted pocket between two beta-sheets to accomplish methyl transfer. Our studies provide the first glimpses, to our knowledge, into the architecture of the nsp14-nsp10 complex involved in RNA viral proofreading.
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