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. 2020 May 14;181(4):894-904.e9.
doi: 10.1016/j.cell.2020.03.045. Epub 2020 Apr 9.

Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2

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Free PMC article

Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2

Qihui Wang et al. Cell. .
Free PMC article

Abstract

The recent emergence of a novel coronavirus (SARS-CoV-2) in China has caused significant public health concerns. Recently, ACE2 was reported as an entry receptor for SARS-CoV-2. In this study, we present the crystal structure of the C-terminal domain of SARS-CoV-2 (SARS-CoV-2-CTD) spike (S) protein in complex with human ACE2 (hACE2), which reveals a hACE2-binding mode similar overall to that observed for SARS-CoV. However, atomic details at the binding interface demonstrate that key residue substitutions in SARS-CoV-2-CTD slightly strengthen the interaction and lead to higher affinity for receptor binding than SARS-RBD. Additionally, a panel of murine monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against SARS-CoV-S1/receptor-binding domain (RBD) were unable to interact with the SARS-CoV-2 S protein, indicating notable differences in antigenicity between SARS-CoV and SARS-CoV-2. These findings shed light on the viral pathogenesis and provide important structural information regarding development of therapeutic countermeasures against the emerging virus.

Keywords: ACE2; CTD; SARS-CoV-2; crystal structure; immunogenicity; receptor; receptor binding domain.

Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

None
Figure S1
Figure S1
Phylogenetic Analysis of SARS-CoV-2 and Sequence Alignments at the CTD Region, Related to Figures 2 and 3 (A) Schematic representation of the SARS-CoV-2 S protein based on the SARS-CoV S protein. (B) Phylogenetic tree generated using MEGA (Tamura et al., 2013) with the S protein sequences. (C) Phylogenetic tree generated using MEGA (Tamura et al., 2013) with the CTD region. (D) Structure-based sequence alignment. The secondary structure elements were defined based on an ESPript (Robert and Gouet, 2014) algorithm and are labeled based on the SARS-CoV-2-CTD structure reported in this study. Spiral lines indicate α or 310 helices, and arrows represent β strands. The Arabic numerals 1-4 indicate cysteine residues that pair to form disulfide bonds. The red rectangles and blue triangles indicate the residues in the SARS-CoV-2-CTD and the SARS-RBD that interact with hACE2, respectively. Two deletions present in the ZXC21 and ZC45 external subdomains were highlighted with green boxes. The red lines indicate the epitopes recognized by mAb1 or mAb2/3.
Figure 1
Figure 1
SARS-CoV-2-S1 and SARS-CoV-2-CTD Co-localize with hACE2 HEK293T cells were transfected with pEGFP-N1-hACE2 (left panels, hACE2-GFP) or pEGFP-C1-hCD26 (right panels, hCD26-GFP). Twenty-four hours later, the cells were incubated with supernatant containing mFc-tagged SARS-CoV-2-S1 (SARS-CoV-2-S1-mFc), SARS-CoV-2-NTD (SARS-CoV-2-NTD-mFc), SARS-CoV-2-CTD (SARS-CoV-2-CTD-mFc), MERS-RBD (MERS-RBD-mFc), or SARS-RBD (SARS-RBD-mFc) proteins and subsequently incubated with anti-mouse IgG (mIgG) antibody conjugated with A594 (anti-mIgG/A594). Nuclei were stained with DAPI. All images were obtained by confocal microscopy using a Leica SP8 (×100 oil immersion objective lens). The scale bar in each panel indicates 8 μm. The data shown are representative of two independent experiments. See also Figure S2.
Figure S2
Figure S2
Characterization of Binding between SARS-CoV-2 and hACE2 by Flow Cytometry, Related to Figures 1 and 2 (A-C) Supernatant containing the indicated mFc-fusion proteins were incubated with HEK293T cells transiently expressing eGFP-tagged hACE2 (A), hCD26 (B) or hAPN (C), respectively. Anti-mIgG/APC was used to detect the mFc-fusion protein binding to the cells. Culture supernatant of HEK239T cells was used as negative control and marked as NC. For each sample, eGFP positive cells were first gated and then used to analyze fluorescence intensity of APC. (D-F) Supernatant containing SARS-CoV-2-CTD-mFc proteins were pre-incubated with soluble hACE2 (D), hCD26 (E) or hAPC (F) at the indicated concentrations before addition to HEK293T cells transfected with pEGFP-N1-hACE2. mFc-fusion protein binding to HEK293T cells were detected by anti-mIgG/APC. (G-I) Supernatant containing SARS-RBD-mFc proteins were pre-incubated with soluble hACE2 (G), hCD26 (H) or hAPC (I) at the indicated concentrations before addition to HEK293T cells transfected with pEGFP-N1-hACE2. mFc-fusion protein binding to HEK293T cells were detected by anti-mIgG/APC. (J-K) HEK293T cells transfected with pEGFP-N1-hACE2 were pre-incubated with soluble SARS-RBD at the indicated concentration, before the addition of supernatant containing either SARS-CoV-2-CTD-mFc (J) or SARS-RBD-mFc (K). mFc-fusion protein binding to HEK293T cells were detected by anti-mIgG/APC. (L-M) HEK293T cells transfected with pEGFP-N1-hACE2 (WT), or the mutants containing K353A (K353A) or K31A (K31A) were incubated with supernatant containing either SARS-CoV-2-CTD-mFc (L) or SARS-RBD-mFc (M). mFc-fusion protein binding to HEK293T cells were detected by anti-mIgG/APC. All data shown are representative of two independent experiments. The fluorescence signals were monitored by BD FACSCanto and the results were analyzed using FlowJo V10 (https://www.flowjo.com/solutions/flowjo/downloads).
Figure 2
Figure 2
The Complex Structure of SARS-CoV-2-CTD Bound to hACE2 (A) A cartoon representation of the complex structure. The core subdomain and external subdomain in SARS-CoV-2-CTD are colored cyan and orange, respectively. hACE2 subdomain I and II are colored violet and green, respectively. The right panel was obtained by anticlockwise rotation of the left panel along a longitudinal axis. The contacting sites are further delineated in (C)–(E) for the amino acid interaction details. (B) A carton representation of the SARS-CoV-2-CTD structure. The secondary structural elements are labeled according to their occurrence in sequence and location in the subdomains. Specifically, the β strands constituting the core subdomain are labeled with an extra c, whereas the elements in the external subdomain are labeled with an extra prime symbol. The disulfide bonds and N-glycan linked to N343 are shown as sticks and spheres, respectively. (C–E) Key contact sites are marked with the left, middle and right box in (A) and further delineated for interaction details, respectively. The residues involved are shown and labeled. See also Figures S1, S2, and S3 and Table S1.
Figure S3
Figure S3
Representative Electron Density Maps at the Binding Interface, Related to Figure 2 The electron densities of residues at the interaction interface between SARS-CoV-2-CTD and hACE2. The density maps are drawn in gray mesh contoured at 1 sigma. The core and external subdomains are colored cyan and orange, respectively. hACE2 is marked in violet. Residues in hACE2 that interact with the SARS-CoV-2-CTD are highlighted in lemon.
Figure 3
Figure 3
Comparison of the SARS-CoV-2-CTD/hACE2 and SARS-RBD/hACE2 Binding Sites (A) Overall similar receptor binding modes were observed for SARS-CoV-2-CTD and SARS-RBD. Superimposition of the structure of SARS-CoV-2-CTD (external subdomain in orange and core subdomain in cyan) bound to hACE2 (violet) and a complex structure of SARS-RBD (in gray) with hACE2 (yellow) are shown. The loop exhibiting variant conformations is highlighted by a dashed oval. (B) hACE2 displayed in surface view. Residues that interact with the SARS-CoV-2-CTD are marked. (C) hACE2 displayed in surface view. Residues that interact with the SARS-RBD are marked. (D) Residues substitutions in SARS-CoV-2-CTD slightly strengthen the interaction with the receptor compared to the SARS-RBD. The amino acid sequences of the loop specified in (A) were aligned between the SARS-CoV-2-CTD and the SARS-RBD. The numbers show the vdw contacts between the receptor with the indicated SARS-CoV-2-CTD residues (above the sequence) or SARS-RBD residues (below the sequence). Numbers in parentheses indicate the number of potential H-bonds conferred by the indicated residues. The red and blue arrows represent the amino acids that form ionic and aromatic-aromatic interactions with the receptor, respectively. See also Figure S1 and Table S1.
Figure 4
Figure 4
Specific Interactions between SARS-CoV-2-S1 and SARS-CoV-2-CTD with hACE2, Characterized by SPR The indicated mFc-tagged proteins in the supernatant were captured by anti-mIgG antibodies that were immobilized on the chip and subsequently tested for binding with gradient concentrations of hACE2 or hCD26, with the following binding profiles shown. (A) SARS-RBD binding to hACE2. (B) SARS-RBD binding to hCD26. (C) MERS-RBD binding to hACE2. (D) MERS-RBD binding to hCD26. (E) SARS-CoV-2-S1 binding to hACE2. (F) SARS-CoV-2-S1 binding to hCD26. (G) SARS-CoV-2-NTD binding to hACE2. (H) SARS-CoV-2-NTD binding to hCD26. (I) SARS-CoV-2-CTD binding to hACE2. (J) SARS-CoV-2-CTD binding to hCD26. (K) Culture supernatant of HEK293T cells without transfection (NC) binding to hACE2. (L) Culture supernatant of HEK293T cells without transfection (NC) binding to hCD26. The values shown are the mean ± SD of three independent experiments.
Figure 5
Figure 5
Different Antigenicity between the SARS-CoV-2 S and SARS-CoV S Proteins (A and B) HEK293T cells were transfected with pCAGGS plasmids containing Flag-tagged SARS-CoV S (A) or SARS-CoV-2 S (B). The indicated purified murine mAbs were subsequently added to the transfected cells before they were fixed, permeabilized, and stained with anti-Flag/fluorescein isothiocyanate (FITC). (C and D) HEK293T cells were transfected with pCAGGS plasmids expressing Flag-tagged SARS-CoV S. The murine polyclonal sera against SARS-RBD (C) or MERS-RBD (D) were subsequently added to the transfected cells before they were fixed, permeabilized, and stained with anti-Flag/FITC. (E and F) HEK293T cells were transfected with pCAGGS plasmids expressing Flag-tagged SARS-CoV-2 S. The murine polyclonal sera against SARS-RBD (E) or MERS-RBD (F) were subsequently added to the transfected cells before they were fixed, permeabilized, and stained with anti-Flag/FITC. (G) Electrostatic surface view of SARS-CoV-2-CTD. The first panel represents the top view. The others are yielded by rotation of the former panel along a horizontal axis. (H) Electrostatic surface view of SARS-RBD. The first panel represents the top view. The others are yielded by rotation of the former panel along a horizontal axis.

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