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

To investigate the structure of nsp13, we overexpressed the full-length MERS-CoV nsp13 (1-598aa) in High-5 insect cells (Fig 1A) . To verify that the recombinant protein is enzymatically active we first performed ATPase assay. The purified nsp13 exhibited ATPase activity with a turnover number (k cat ) of 2.03 ±0.1 s -1 and the catalytic efficiency (k cat K m -1 ) of 0.32 μM -1 s -1 (Fig 1B) . The ATPase activity of MERS-CoV nsp13 is comparable with that reported for SARS nsp13 [29] . Next, we assessed helicase activity of the recombinant nsp13. Partial RNA duplex containing 5' overhang was fully unwound by the purified nsp13. By contrast, MERS-CoV nsp13 could not unwind RNA duplex containing 3' overhang (the duplex region remained the same as the RNA duplex with 5' overhang). These results confirm that MERS-CoV nsp13 is a unidirectional helicase with the unwinding polarity of 5'-to-3' (Fig 1C, left) . Mutant with E375Q within Walker B failed to unwind the RNA substrate with 5' overhang clearly indicating that the helicase activity of MERS-CoV nsp13 is dependent on ATP hydrolysis (Fig 1C, left) . MERS-CoV nsp13 was able to hydrolyze different NTPs and dNTPs to support the unwinding of RNA substrate, with a clear preference towards ATP (Fig 1C, right) . Our results are consistent with the recent enzymatic characterization of the MERS-CoV nsp13 expressed in bacteria [30] .