title: Structural basis and functional analysis of the SARS coronavirus nsp14-nsp10 complex.
abstract: 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 β-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 β-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.
authors.affiliation: Laboratory of Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China;
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Ma
authors.affiliation: National Center for Protein Science Shanghai, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China;
authors.forename: Lijie
authors.initials: L
authors.lastname: Wu
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China;
authors.forename: Neil
authors.initials: N
authors.lastname: Shaw
authors.affiliation: Laboratory of Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China;
authors.forename: Yan
authors.initials: Y
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Biotherapy and Cancer Center, Sichuan University, Chengdu, Sichuan 610041, China; Collaborative Innovation Center for Biotherapy Chengdu, Sichuan 610041, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China;
authors.forename: Yuna
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Laboratory of Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China;
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Lou
authors.affiliation: Laboratory of Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China;
authors.forename: Liming
authors.initials: L
authors.lastname: Yan
authors.affiliation: National Center for Protein Science Shanghai, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; raozh@mail.tsinghua.edu.cn rzhang@sun5.ibp.ac.cn.
authors.forename: Rongguang
authors.initials: R
authors.lastname: Zhang
authors.affiliation: Laboratory of Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China; National Center for Protein Science Shanghai, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; State Key Laboratory of Biotherapy and Cancer Center, Sichuan University, Chengdu, Sichuan 610041, China; Collaborative Innovation Center for Biotherapy Chengdu, Sichuan 610041, China raozh@mail.tsinghua.edu.cn rzhang@sun5.ibp.ac.cn.
authors.forename: Zihe
authors.initials: Z
authors.lastname: Rao
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism; D005095:Exoribonucleases / Q000737:chemistry*; D008024:Ligands; D008780:Methyltransferases / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D045473:Severe acute respiratory syndrome-related coronavirus / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D014779:Virus Replication / Q000235:genetics; D016335:Zinc Fingers
chemical_list: D008024:Ligands; C558878:Nsp10 protein, SARS virus; D012333:RNA, Messenger; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases; C525596:nsp14 protein, SARS coronavirus; D005095:Exoribonucleases
keywords: CoV; exoribonuclease; methyltransferase; nsp14; proofreading