Structures of theMiddle East respiratory syndrome coronavirus3C-like protease reveal insights into substrate specificity

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic virus that causes severe respiratory illness accompanied by multiorgan 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.

Structural basis for the dimerization and substrate recognition specificity of porcine epidemic diarrhea virus 3C-like protease

“…Each monomer features three domains: domain I (residues 1-97), domain II (residues 98-186), and domain III (residues 202-298). Each of the first two domains exhibits an antiparallel β-barrel structure, which is similar to the 3CL pro s from other coronaviruses (Anand et al, 2002;Needle et al, 2015;Yang et al, 2003). Domain III consists of five α helices and is connected to domain II by a long loop (residues 187-197, loop A in Fig.…”

Identification and evaluation of potent Middle East respiratory syndrome coronavirus (MERS-CoV) 3CL Pro inhibitors

“…Then, the simulation was done by Discovery Studio (Accelrys Inc., San Diego, CA). A Cys at subsite S1 of 3CL pro acts as a nucleophile to cleave substrates by attacking carbonyl carbon of the amide bond between the conserved Gln at P1 and the small amino acids such as Ser, Ala or Gly at P1' (Fan et al, 2004;Needle et al, 2015). Our modelling shows that the g-sulfur of Cys148 forms a covalent bond with the 6d aldehyde carbon and the resulting oxyanion is stabilized by His41 (Fig.…”

Characteristics of flavonoids as potent MERS‐CoV 3C‐like protease inhibitors

“…The homodimeric form of 3CLpro is active in the presence of substrates. The crystal structure of 3CLpro showed that each monomer is composed of three structural domains: domains I and II form a chymotrypsinlike architecture with catalytic cysteine and are connected to a third C-terminal domain via a long loop (Neddle, Lountos, & Waugh, 2015). In the proteolytic site, all 3CLpros prefer glutamine at P1 position and leucine, basic residues, small hydrophobic residues at P2, P3 and P4 positions, respectively (Chuck, Chow, Wan, & Wong, 2011).…”