Structures of the Middle East respiratory syndrome coronavirus 3C-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 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 (3 CL(pro)). Since 3 CL(pro) is essential for viral replication, it is a leading candidate for therapeutic intervention. To accelerate the development of 3 CL(pro) inhibitors, three crystal structures of a catalytically inactive variant (C148A) of the MERS-CoV 3 CL(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 3 CL(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 3 CL(pro) enzyme.

Keywords: 3CLpro; MERS-CoV; coronavirus; main protease.

Figure 1

The catalytically inactive MERS-CoV 3CL^pro C148A homodimer as found in crystal form I. Protomer A is colored green and protomer B red. The residues forming the catalytic dyad are depicted as blue spheres.

Figure 2

(a) The C-terminal residues of protomer D (crystal form II), corresponding to the P6–P1 autoprocessed site of the mature enzyme fitted to the mF _o − DF _c electron-density maps shown (contour level of 3.0σ, green; 1.55 Å resolution) after a round of refinement with the C-­terminal residues omitted from the model. (b) Illustration of the binding of the C-terminal tail (spheres) of protomer D (magenta ribbons) to the homodimer formed by protomer A (gray surface) and protomer B (cyan surface).

Figure 3

(a) Stereoview of the superimposed homodimers of MERS-CoV 3CL^pro (crystal form II, green ribbons) and BatCoV-HKU4 (PDB entry 2yna, red ribbons). (b) Stereoview of the superimposed homodimers of MERS-CoV 3CL^pro and SAR-CoV 3CL^pro (PDB entry 1uk3, red ribbons; Yang et al., 2003 ▶).

Figure 4

Sequence alignment of CoV 3CL^pro enzymes from MERS-CoV, SARS-CoV, Tylonycteris bat coronavirus HKU4, Human coronavirus HKU1, Human coronavirus OC43, Human coronavirus NL63 and Human coronavirus 229E. Sequences were aligned using T-Coffee (Notredame et al., 2000 ▶) and the figure was prepared with ESPript3 (Robert & Gouet, 2014 ▶). The residues forming the catalytic dyad are highlighted with asterisks.

Figure 5

(a) Stereoview of the hydrogen-bonding interactions (within 3.2 Å) between the C-terminal residues 301–306 of MERS-CoV 3CL^pro protomer D (crystal form II, C atoms in green) and the active site of protomer A (C atoms in gray). Residue Ser1 (C atoms in yellow) is from protomer B of the homodimer. (b) Stereoview of the active-site residues from protomer A of the free enzyme form (crystal form I, C atoms in magenta) superimposed onto the active site of product-bound protomer A (crystal form II, C atoms in gray).

Figure 6

(a) Stereoview of the superimposed structures of MERS-CoV 3CL^pro crystal form III protomer A (green ribbons) and protomer C (magenta ribbons). (b, c) Surface representations of protomer A (b) and protomer C (c) with domains I and II colored gray, the linker loop (residues 188–204) cyan, domain III magenta, the oxyanion loop (residues 143–148) blue and the S1 binding pocket green.

Figure 7

(a) Stereoview of the structure of MERS-CoV 3CL^pro protomer C (crystal form III, magenta ribbons) superimposed on the structure of IBV-CoV 3CL^pro protomer C (PDB entry 2q6d, red ribbons; Xue et al., 2008 ▶). (b) Stereoview of the structure of MERS-CoV 3CL^pro protomer C (crystal form III, magenta ribbons) superimposed on the structure of the SARS-CoV 3CL^pro G11A monomer (PDB entry 2pwx, cyan ribbons; Chen et al., 2008 ▶).