Crystal structure of the receptor-binding domain from newly emerged Middle East respiratory syndrome coronavirus

Abstract

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 77 people, with a fatality rate of more than 50%. Alarmingly, the virus demonstrates the capability of human-to-human transmission, raising the possibility of global spread and endangering world health and economy. Here we have identified the receptor-binding domain (RBD) from the MERS-CoV spike protein and determined its crystal structure. This study also presents a structural comparison of MERS-CoV RBD with other coronavirus RBDs, successfully positioning MERS-CoV on the landscape of coronavirus evolution and providing insights into receptor binding by MERS-CoV. Furthermore, we found that MERS-CoV RBD functions as an effective entry inhibitor of MERS-CoV. The identified MERS-CoV RBD may also serve as a potential candidate for MERS-CoV subunit vaccines. Overall, this study enhances our understanding of the evolution of coronavirus RBDs, provides insights into receptor recognition by MERS-CoV, and may help control the transmission of MERS-CoV in humans.

Fig 1

Crystal structure of the MERS-CoV S1 C-domain. (A) Domain structure of MERS-CoV S1 subunit that contains an N-terminal domain (NTD) and a C-domain. The boundaries of the C-domain and proposed receptor-binding motif (RBM) of MERS-CoV were determined by sequence and structural comparisons with the SARS-CoV S1 subunit (see Fig. 2). (B) Sequence and secondary structure of the MERS-CoV C-domain. Helices are drawn as cylinders, and strands are drawn as arrows. The disordered region is shown as a dashed line. (C) Crystal structure of the MERS-CoV C-domain. The core structure is in cyan, and the proposed RBM region is in red.

Fig 2

Sequence and structural comparisons of MERS-CoV and SARS-CoV S1 C-domains. (A) Sequence alignment of MERS-CoV and SARS-CoV C-domains. Residues corresponding to C-domain core structures are in cyan, and residues corresponding to the proposed RBM region are in red. Cysteine residues are highlighted. Asterisks indicate positions that have fully conserved residues; colons indicate positions that have strongly conserved residues; periods indicate positions that have weakly conserved residues. (B) Crystal structure of MERS-CoV C-domain. The secondary structures are colored in the same way as the corresponding sequence in panel A. Disulfide bond-linked cysteine residues are shown as sticks in yellow, and N-linked glycans are shown as sticks in green. (C) Crystal structures of SARS-CoV C-domain (PDB 2AJF). Two disordered loops are drawn as dashed lines. Although cysteines 377 and 511 are disordered in this structure, they were shown to form a disulfide bond in another study (46).

Fig 3

Interactions between the MERS-CoV S1 C-domain and human DPP4. (A) Dose-dependent inhibition of MERS-CoV infection by the MERS-CoV C-domain. Vero cells were treated with increasing concentrations of the MERS-CoV C-domain prior to infection. Virus titers were determined by plaque assay and are shown as PFU/ml. (B) Inhibition of MERS-CoV spike-pseudotyped retrovirus infection by MERS-CoV C-domain. MERS-CoV spike-pseudotyped MLV and VSV-G-pseudotyped MLV were mixed with the MERS-CoV C-domain and BSA, respectively. HEK293T cells transiently expressing human DPP4 were infected with different amount of pseudotyped virus alone or its mixture with proteins. At 48 h postinfection, cells were lysed and β-galactosidase activities were measured. The β-galactosidase activity of cell lysate infected with 10 μl pseudotyped virus alone was taken as 100%. Values are means and standard errors of the means (SEM). (C) Pulldown of cell surface DPP4 by MERS-CoV C-domain. 293T cells were transfected with either empty vector or vector containing DPP4-HA gene. At 48 h posttransfection, cells were harvested and lysed. Cell lysate was mixed with the MERS-CoV C-domain containing a C-terminal His₆ tag. The C-domain/DPP4 complex was then precipitated with Ni-NTA beads.

Fig 4

Comparison of receptor-binding mechanisms of coronavirus S1 C-domains. These C-domains include HCoV-NL63 RBD complexed with human ACE2 (PDB 3KBH), PRCV RBD complexed with porcine APN (4F5C), and SARS-CoV RBD complexed with human ACE2 (2AJF). The arrow represents interaction between the MERS-CoV RBD and human DPP4. RBMs and core structures of coronavirus C-domains are in red and cyan, respectively, and receptors are in green.

Fig 5

Comparison of tertiary structures of coronavirus S1 C-domains. (Top) Tertiary structures of coronavirus C-domains (PDB IDs are the same as in Fig. 4). (Bottom) Schematic illustration of the structural topologies of coronavirus C-domains. β-strands are depicted as arrows and α-helices as cylinders. The secondary structures of all of the coronavirus C-domains are colored and numbered in the same way as for the HCoV-NL63 C-domain.

Fig 6

Proposed evolution of the structures and functions of coronavirus S1 C-domains.