Structure of the multiple functional domains from coronavirus nonstructural protein 3

Mengxia Li, Gang Ye, Yu Si, Zhou Shen, Zhu Liu, Yuejun Shi, Shaobo Xiao, Zhen F. Fu, Guiqing Peng

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


Coronaviruses (CoVs) are potential pandemic pathogens that can infect a variety of hosts and cause respiratory, enteric, hepatic and neurological diseases. Nonstructural protein 3 (nsp3), an essential component of the replication/transcription complex, is one of the most important antiviral targets. Here, we report the first crystal structure of multiple functional domains from porcine delta-coronavirus (PDCoV) nsp3, including the macro domain (Macro), ubiquitin-like domain 2 (Ubl2) and papain-like protease (PLpro) catalytic domain. In the asymmetric unit, two of the subunits form the head-to-tail homodimer with an interaction interface between Macro and PLpro. However, PDCoV Macro-Ubl2-PLpro mainly exists as a monomer in solution. Then, we conducted fluorescent resonance energy transfer-based protease assays and found that PDCoV PLpro can cleave a peptide by mimicking the cognate nsp2/nsp3 cleavage site in peptide substrates and exhibits deubiquitinating and de-interferon stimulated gene(deISGylating) activities by hydrolysing ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) and ISG15-AMC substrates. Moreover, the deletion of Macro or Macro-Ubl2 decreased the enzyme activity of PLpro, indicating that Macro and Ubl2 play important roles in maintaining the stability of the PLpro domain. Two active sites of PLpro, Cys260 and His398, were determined; unexpectedly, the conserved site Asp412 was not the third active site. Furthermore, the motif “NGYDT” (amino acids 409–413) was important for stabilizing the enzyme activity of PLpro, and the N409A mutant significantly decreased the enzyme activity of PLpro. These results provide novel insights into the replication mechanism of CoV and new clues for future drug design.

Original languageEnglish (US)
Pages (from-to)66-80
Number of pages15
JournalEmerging Microbes and Infections
Issue number1
StatePublished - 2021
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd.


  • Multiple functional domains
  • enzyme activity
  • macro domain
  • papain-like protein
  • ubiquitin-like domain


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