Crystal structure of Middle East respiratory syndrome coronavirus helicase

Hao, Wei; Wojdyla, Justyna Aleksandra; Zhao, Rong; Han, Ruiyun; Das, Rajat; Zlatev, Ivan; Manoharan, Muthiah; Wang, Meitian; Cui, Sheng

Structural comparisons of the individual domains of Upf1-like helicases using Dali server showed that the CH of MERS-CoV nsp13 is structurally closer to the CH of Upf1 than to the ZBD of EAV nsp10. In accordance with previously published results, the structure of the helicase cores is well conserved in all three Upf1-like helicases (Table 2) . However, some subtle differences could also be identified. Firstly, while the helicase cores of MERS-CoV nsp13 and Upf1 have similar size, the helicase core of EAV nsp10 is more compact. MERS-CoV nsp13 contains seven and five parallel β-strands in RecA1 and RecA2 domains, respectively, Upf1 has seven and six β-strands in the RecA1 and RecA2 domain, respectively, whereas EAV nsp10 has only five and four β-strands in the RecA like domains. Secondly, while three Upf1 like helicases all contain the 1B domain with the β-barrel fold, only Upf1 has a helical 1C insertion in RecA1 domain. The equivalent 1C insertion is missing in both EAV nsp10 [25] and MERS-CoV nsp13 (Fig 4D and 4E) . Thirdly, CoVs nsp13 does not contain a C-terminal domain homologous to the C-terminal portion of EAV nsp10 (C-terminal 65 residues), which was shown to regulate ATPase and helicase activities [25] . The sequence conservation analysis showed that the C-terminal domain of arteriviruses and CoVs is indeed poorly conserved [25] . Our crystallographic study provides the complete structure of the extreme C-terminal region of MERS-CoV nsp13, which shows that the C-terminus is an integral part of the RecA2 domain. Thus, we conclude that the C-terminal regulatory domain outside SF1 helicase core is completely missing in MERS-CoV nsp13 and other CoVs helicases. Previous mutagenesis studies of CoVs nsp13 have identified a collection of residues essential to the activity of nsp13. Because nsp13 helicase is highly conserved among CoVs, the crystal structure of MERS-CoV nsp13 can provide three-dimensional information to understand previous phenotypes of nsp13 mutants. It has been shown that mutations of the conserved Cys/His at ZBD of 229E-CoV nsp13 interfered with its ATPase activity [32] . Ala or Arg replacement of C5003, C5021, C5024 and H5028 abolished or reduced the ATPase activity of 229E-CoV nsp13. Our crystal structure of MERS-CoV nsp13 confirms that these residues, corresponding to C8, C26, C29 and H33 of MERS-CoV nsp13 (Fig 4C) are coordinating Zn1 and Zn2. The loss of ATPase activity caused by Cys/His substitutes can be attributed to the disruption of zinc-binding and the integrity of the CH domain. Unexpectedly, only C5050A mutant retained significant ATPase activity (~60%). Based on the comparison with corresponding C55 of MERS-CoV nsp13, C5050 of 229E-CoV coordinates Zn3 of the ZBD. Zn3 is the most distantly located zinc from the helicase core (6.4Å away from Zn2; Fig 1A) , and the ZBD/CH is tightly attached to the helicase core in CoV nsp13, it is therefore unsurprising that the impaired binding of Zn3 has the minimal effect on the enzymatic activity of nsp13 [32] . This hypothesis is supported by the mutagenesis study of EAV nsp10 [25, 32] , which also showed that while mutant H2414A (ligand for Zn3) retained residual ATPase/ helicase activity and wild-type-level nucleic acid binding activity, the activities of mutants C2395A (ligand for Zn1) and H2399A (ligand for Zn2) was completely abolished.