Structures of the Middle East respiratory syndrome coronavirus 3C‐like protease reveal insights into substrate specificity

Middle East respiratory syndrome coronavirus (MERS‐CoV) is a highly pathogenic virus that causes severe respiratory illness accompanied by multi‐organ dysfunction, resulting in a case fatality rate of approximately 40%. As found in other coronaviruses, the majority of the positive‐stranded RNA MERS‐CoV genome is translated into two polyproteins, one created by a ribosomal frameshift, that are cleaved at three sites by a papain‐like protease and at 11 sites by a 3C‐like protease (3CL(pro)). Since 3CL(pro) is essential for viral replication, it is a leading candidate for therapeutic intervention. To accelerate the development of 3CL(pro) inhibitors, three crystal structures of a catalytically inactive variant (C148A) of the MERS‐CoV 3CL(pro) enzyme were determined. The aim was to co‐crystallize the inactive enzyme with a peptide substrate. Fortuitously, however, in two of the structures the C‐terminus of one protomer is bound in the active site of a neighboring molecule, providing a snapshot of an enzyme–product complex. In the third structure, two of the three protomers in the asymmetric unit form a homodimer similar to that of SARS‐CoV 3CL(pro); however, the third protomer adopts a radically different conformation that is likely to correspond to a crystallographic monomer, indicative of substantial structural plasticity in the enzyme. The results presented here provide a foundation for the structure‐based design of small‐molecule inhibitors of the MERS‐CoV 3CL(pro) enzyme.