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.

In this study, by utilizing immunostaining and flow cytometry assays, we first identify the S1 CTD (SARS-CoV-2-CTD) as the key region in SARS-CoV-2 that interacts with the hACE2 receptor. We subsequently solved a 2.5-Å crystal structure of SARS-CoV-2-CTD in complex with hACE2, which reveals a receptor-binding mode similar overall to that observed for the SARS-CoV RBD (SARS-RBD). However, SARS-CoV-2-CTD forms more atomic interactions with hACE2 than SARS-RBD, which correlates with data showing higher affinity for receptor binding. Notably, a panel of monoclonal antibodies (mAbs) as well as murine polyclonal antisera against SARS-S1/RBD were unable to bind to the SARS-CoV-2 S protein, indicating notable differences in antigenicity between SARS-CoV and SARS-CoV-2, suggesting that the previously developed SARS-RBD-based vaccine candidates are unlikely to be of any clinical benefit for SARS-CoV-2 prophylaxis. Taken together, these data shed light on viral entry and pathogenesis and will hopefully inspire new targeted treatments for this emerging pathogen.

In CoVs, the entry process is mediated by the envelope-embedded surface-located spike (S) glycoprotein (). This S protein would, in most cases, be cleaved by host proteases into the S1 and S2 subunits, which are responsible for receptor recognition and membrane fusion, respectively (). S1 can be further divided into an N-terminal domain (NTD) and a C-terminal domain (CTD), both of which can function as a receptor-binding entity (e.g., SARS-CoV and MERS-CoV utilize the S1 CTD to recognize the receptor (also called receptor binding domain [RBD]) (), whereas mouse hepatitis CoV engages the receptor with its S1 NTD ()). The region in SARS-CoV-2 S protein that is responsible for hACE2 interaction remains unknown.

Virus infections initiate with binding of viral particles to host surface cellular receptors. Receptor recognition is therefore an important determinant of the cell and tissue tropism of a virus. In addition, the gain of function of a virus to bind to the receptor counterparts in other species is also a prerequisite for inter-species transmission (). Interestingly, with the exception of HCoV-OC43 and HKU1, both of which have been shown to engage sugars for cell attachment (), the other human CoVs recognize proteinaceous peptidases as receptors. HCoV-229E binds to human aminopeptidase N (hAPN) (), and MERS-CoV interacts with human dipeptidyl peptidase 4 (hDPP4 or hCD26) (). Although they belong to different genera, SARS-CoV and hCoV-NL63 interact with human angiotensin-converting enzyme 2 (hACE2) for virus entry (). After the outbreak of COVID-19, Chinese scientists promptly determined that SARS-CoV-2 also utilizes hACE2 for cell entry ().

SARS-CoV-2 is the seventh coronavirus that is known to cause human disease. Coronaviruses (CoVs) are a group of large and enveloped viruses with positive-sense, single-stranded RNA genomes (). The viruses can be classified into four genera: alpha, beta, gamma, and deltaCoVs ( https://talk.ictvonline.org/ ). Previously identified human CoVs that cause human disease include the alphaCoVs hCoV-NL63 and hCoV-229E and the betaCoVs HCoV-OC43, HKU1, severe acute respiratory syndrome CoV (SARS-CoV), and Middle East respiratory syndrome CoV (MERS-CoV) (). Both alphaCoVs and the betaCoVs HCoV-OC43 and HKU1 cause self-limiting common cold-like illnesses (). However, SARS-CoV and MERS-CoV infection can result in life-threatening disease and have pandemic potential. During 2002–2003, SARS-CoV initially emerged in China and swiftly spread to other parts of the world, causing more than 8,000 infections and approximately 800 related deaths worldwide (). In 2012, MERS-CoV was first identified in the Middle East and then spread to other countries (). As of November 2019, a total of 2,494 MERS cases with 858 related deaths have been recorded in 27 countries globally ( https://www.who.int/emergencies/mers-cov/en/ ). Notably, new cases of MERS-CoV infecting humans are still being reported ( https://www.who.int/csr/don/archive/disease/coronavirus_infections/en/ ). SARS-CoV and MERS-CoV are zoonotic pathogens originating from animals. Detailed investigations indicate that SARS-CoV is transmitted from civet cats to humans and MERS-CoV from dromedary camels to humans (). The source of SARS-CoV-2, however, is still under investigation but linked to a wet animal market ().

The 2019-nCoV Outbreak Joint Field Epidemiology Investigation Team Notes from the Field: An Outbreak of NCIP (2019-nCoV) Infection in China - Wuhan, Hubei Province, 2019–2020.

Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China.

Emerging and re-emerging viruses are a significant threat to global public health (). Since the end of 2019, Chinese authorities have reported a cluster of human pneumonia cases in Wuhan City, China (), and the disease was designated coronavirus disease 2019 (COVID-19). These cases showed symptoms such as fever and dyspnea and were diagnosed as viral pneumonia (). Whole-genome sequencing results showed that the causative agent was a novel coronavirus that was initially named 2019-nCoV by the World Health Organization (WHO) (). Later, the International Committee on Taxonomy of Viruses (ICTV) officially designated the virus SARS-CoV-2 (), although many virologists argue that HCoV-19 is more appropriate (). As of February 24, 2020, 79,331 laboratory-confirmed cases have been reported to the WHO globally, with 77,262 cases in China, including 2,595 deaths ( https://www.who.int/ ). In addition, 29 other countries have confirmed imported cases of SARS-CoV-2 infection ( https://www.who.int/ ), raising great public health concerns worldwide.

Coronaviridae Study Group of the International Committee on Taxonomy of Viruses The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2.

In comparison with a limited number of mAbs, polyclonal antibodies provide a more comprehensive view on potential epitope differences. In light of the determinant role of SARS-RBD and MERS-RBD in receptor recognition (), the majority of neutralizing antibodies have been shown to target the RBD, exerting neutralization activity by disrupting virus/receptor engagement (). We therefore further prepared murine polyclonal antibodies against SARS-RBD and MERS-RBD. These two viral RBDs share very limited sequence identity and exhibit distinct structural characteristics in the RBD external subdomain that mediates receptor binding (). In the positive control, anti-SARS-RBD antibodies, but not anti-MERS-RBD antibodies, potently bound to cells expressing SARS-CoV S, as expected ( Figures 5 C and 5D). Nonetheless, neither of the antibody preparations bound to SARS-CoV-2 S ( Figures 5 E and 5F). In agreement with this observation, although SARS-CoV-2-CTD is structurally similar to the SARS-RBD structures ( Figure 3 ), the electrostatic surface potential maps of these proteins were different ( Figure 5 G and 5H), which might explain the differing immunogenicity of the two ligands. Therefore, the results highlight distinct epitope features between SARS-RBD and SARS-CoV-2-CTD, although both can engage hACE2.

To conclude, we set out to investigate the epitope features of SARS-CoV-2 S by using a panel of murine mAbs directed against SARS-CoV S, including the B30A38, A50A1A1, and C31A12 antibodies, which recognize SARS-CoV S1, and mAbs 1–3, which recognize the SARS-RBD ( Figure S1 D;). Using flow cytometry, all six mAbs were observed to effectively bind to cells expressing SARS-CoV S. None of the mAbs, however, interacted with SARS-CoV-2 S ( Figures 5 A and 5B ).

(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.

(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.

(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.

(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.

(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).

Preparation and characterization of monoclonal antibodies against S1 domain at N-terminal residues 249 to 667 of SARS-associated coronavirus S1 protein.

In light of the increased atomic interactions between hACE2 with the SARS-CoV-2-CTD compared with the SARS-RBD, we speculated that the former should bind to the receptor with stronger affinity than the latter. To test this hypothesis, we performed real-time surface plasmon resonance (SPR) assays. The mFc-tagged S-domain proteins were captured by anti-mouse IgG (mIgG) antibodies that were immobilized on the chip and tested for binding with gradient concentrations of the soluble ectodomain proteins of hACE2 and hCD26. As assay controls, SARS-RBD and MERS-RBD were found to readily interact with their respective canonical receptors ( Figures 4 A and 4D ). SARS-CoV-2-S1 and SARS-CoV-2-CTD bound to hACE2 but not to hCD26 ( Figures 4 E, 4F, 4I, and 4J). The recorded binding profiles revealed typical slow-on/slow-off kinetics, as observed with the SARS-CoV and MERS-CoV proteins. The equilibrium dissociation constants (K) of SARS-CoV-2-S1 and SARS-CoV-2-CTD binding to hACE2 were calculated to be 94.6 ± 6.5 nM and 133.3 ± 5.6 nM, respectively. These values represent ~4-fold higher binding affinities than that observed for the SARS-RBD engaging the same receptor, which was determined to be 408.7 ± 11.1 nM ( Figure 4 ). Taken together, the increased atomic interactions between the hACE2 and SARS-CoV-2-CTD binding region leads to ~4-fold higher binding affinity compared with the SARS-RBD.

The values shown are the mean ± SD of three independent experiments.

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.

The Interaction between SARS-CoV-2-CTD and hACE2 Is Specific and Displays 4-Fold Stronger Affinity Compared with the SARS-RBD

Notably, the most variable loop (β1’/β2′ loop) contributes substantially more vdw contacts in SARS-CoV-2-CTD than for the SARS-RBD (115 versus 53) ( Figure 3 D; Table S1 ). Specifically, F486 in SARS-CoV-2, instead of I472 in SARS-RBD, forms strong aromatic-aromatic interactions with hACE2 Y83, and E484 in the SARS-CoV-2-CTD, instead of P470 in the SARS-RBD, forms ionic interactions with K31 ( Figure 3 D).

The overall structure of the SARS-CoV-2-CTD/hACE2 complex is very similar to the previously reported structure of SARS-RBD bound to the same receptor with an RMSD of 0.431 Å for 669 equivalent Cα atoms ( Figures 3 A–3C). Consistent with this high degree of similarity, the soluble SARS-RBD blocks the interaction between the SARS-CoV-2 ligand with hACE2 in a concentration-dependent manner ( Figures S2 J and S2K). Further detailed comparison of the receptor binding interface between the two viruses reveals that, among the 24 residues in hACE2 that make vdw contacts with either CTD, 15 amino acids display more contacts with the SARS-CoV-2-CTD ( Table 2 ). The SARS-CoV-2-CTD binding interface also has more residues than SARS-RBD (21 versus 17) that directly interact with hACE2, forming more vdw contacts (288 versus 213) as well as H-bonds (16 versus 11) ( Tables 2 and S1 ). Consistently, SARS-CoV-2-CTD in complex with hACE2 buries larger surface areas than SARS-RBD (1773 Åversus 1686 Å).

SARS-CoV-2-CTD exhibits significant structural homology to its SARS-CoV homolog, in agreement with high sequence identity between the two molecules (~73.9%) ( Figure S1 C). Superimposition of the SARS-CoV-2-CTD structure onto a previously reported SARS-RBD structure (PDB: 2GHV ) revealed a root-mean-square deviation (RMSD) of 0.475 Å for 128 equivalent Cα atoms ( Figure 3 A). In comparison with the SARS-RBD, the majority of the secondary structure elements are well superimposed in SARS-CoV-2-CTD, with the exception of the β1′/β2′ loop, which showed the most sequence variation between the two ligands ( Figures 3 A and S1 D).

(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.

(C) hACE2 displayed in surface view. Residues that interact with the SARS-RBD are marked.

(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.

Further analysis was performed to identify key residues involved in complex formation. Amino acids located within the van der Waals (vdw) contact distance (4.5-Å-resolution cutoff) between the viral ligand and receptor were selected ( Table 2 ), and a series of hydrophilic residues located along the interface were found to form a solid network of H-bond and salt bridge interactions ( Figure 2 ). These strong polar contacts include the SARS-CoV-2-CTD residue A475 interacting with hACE2 residue S19, N487 with Q24 ( Figures 2 C and S3 A), E484 with K31, and Y453 with H34 ( Figures 2 D and S3 B). Residue K417, located in helix α3 of the CTD core subdomain, was shown to contribute ionic interactions with hACE2 D30 ( Figures 2 D and S3 B). Notably, the bulged loops in SARS-CoV-2-CTD, the α1’/β1’ loop and β2′/η1’ loop, properly position several residues (G446, Y449, G496, Q498, T500, and G502) in close proximity with hACE2 amino acids D38, Y41, Q42, K353, and D355, forming a concentration of H-bonds ( Figures 2 E and S3 C). Further virus-receptor contacts include SARS-CoV-2-CTD Y489 and F486 packing against hACE2 residues F28, L79, M82, and Y83, forming a small patch of hydrophobic interactions at the interface ( Figures 2 C and S3 A). Overall, the virus-receptor engagement is dominated by polar contacts mediated by the hydrophilic residues. In support of this hypothesis, a single K353A mutation was sufficient to abolish these interactions ( Figure S2 L).

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.

The numbers in parentheses of hACE2 residues represent the number of vdw contacts between the indicated residue with SARS-CoV-2-CTD (the former) and SARS-RBD (the latter). The numbers in parentheses of either ligand residues represent the numbers of wdw contacts the indicated residues conferred. The numbers with underline suggest numbers of potential H-bonds between the pairs of residues. wdw contact was analyzed at a cutoff of 4.5 Å and H-bonds at a cutoff of 3.5 Å. See also Table S1

In the complex structure, the SARS-CoV-2-CTD contains 195 consecutive density-traceable residues spanning T333 to P527 together with N-linked glycosylation at N343. Similar to other reported betaCoV CTD structures, this protein also exhibits two structural domains (). One is the conserved core subdomain with five antiparallel β strands and a conserved disulfide bond between βc2 and βc4 ( Figures 2 B and S1 D). The other is the external subdomain, which is dominated by a disulfide bond-stabilized flexible loop that connects two small β strands. The complex structure data show that SARS-CoV-2-CTD utilizes its external subdomain to recognize subdomain I in the hACE2 NTD ( Figure 2 A;).

We then attempted to study the structural basis of the virus-receptor interaction. We prepared the SARS-CoV-2-CTD/hACE2 complex by in vitro mixture of the two proteins and isolated complexes via size exclusion chromatography. The complex structure was solved to 2.5-Å resolution ( Table 1 ), with one SARS-CoV-2-CTD binding to a single hACE2 molecule in the asymmetric unit. For hACE2, clear electron densities could be traced for 596 residues from S19 to A614 of the N-terminal peptidase domain as well as glycans N-linked to residues 53, 90, and 322 ( Figure 2 A).

(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.

(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.

(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.

We further tested binding of the viral proteins to cell-surface hACE2 via flow cytometry. Consistently, SARS-CoV-2-S1 and SARS-CoV-2-CTD, but not SARS-CoV-2-NTD, showed strong affinity for hACE2 ( Figure S2 A). None of the novel CoV proteins interacted with hCD26 or hAPN ( Figures S2 B and S2C). In addition, soluble hACE2, but not hCD26 or hAPN, was shown to inhibit the interaction between viral proteins, with cells expressing hACE2 in a dose-dependent manner ( Figures S2 D–S2I). Taken together, these results clearly demonstrate that SARS-CoV-2 is capable of binding, via the viral CTD, to hACE2.

The fluorescence signals were monitored by BD FACSCanto and the results were analyzed using FlowJo V10 ( https://www.flowjo.com/solutions/flowjo/downloads ).

(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.

(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.

(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.

(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.

(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.

Characterization of Binding between SARS-CoV-2 and hACE2 by Flow Cytometry, Related to Figures 1 and 2

We prepared a series of mouse Fc (mFc)-fused SARS-CoV-2 S protein preparations, including S1 (SARS-CoV-2-S1), the NTD (SARS-CoV-2-NTD), and the CTD, and subsequently visualized their binding to GFP-tagged hACE2 expressed on the cell surface via confocal fluorescence microscopy. As a control, we also prepared the Fc fusion proteins for SARS-RBD and MERS-RBD and tested these in parallel with the SARS-CoV-2 proteins. As expected, SARS-RBD showed co-localization with hACE2 and MERS-RBD with hCD26. For the novel CoV proteins, SARS-CoV-2-S1 and SARS-CoV-2-CTD co-localized with hACE2 on the cell surface. The SARS-CoV-2-NTD protein, however, was incapable of binding to hACE2. In addition, none of the SARS-CoV-2 proteins interacted with hCD26 ( Figure 1 ).

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.

Through bioinformatics analysis, the SARS-CoV-2 S protein has been shown to display characteristic CoV S features, including a S1 region containing the NTD and CTD, S2, a transmembrane region, and a short cytoplasmic domain ( Figure S1 A). Phylogenetic studies reveal that SARS-CoV-2 belongs to a group containing SARS-CoV as well as two bat-derived SARS-like viruses, ZC45 and ZCX21 ( Figures S1 B–S1D). Recently, hACE2 was reported to be the receptor of SARS-CoV-2 (). Because SARS-CoV utilizes its S1 CTD, otherwise known as the RBD, to recognize the same receptor, we decided to test whether the CTD in SARS-CoV-2 is also the key region for interaction with its receptor hACE2.

(D) Structure-based sequence alignment. The secondary structure elements were defined based on an ESPript () algorithm and are labeled based on the SARS-CoV-2-CTD structure reported in this study. Spiral lines indicate α or 3helices, 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.

Phylogenetic Analysis of SARS-CoV-2 and Sequence Alignments at the CTD Region, Related to Figures 2 and 3

Discussion

Hoffmann et al., 2020 Hoffmann M.

Kleine-Weber H.

Schroeder S.

Krüger N.

Herrler T.

Erichsen S.

Schiergens T.S.

Herrler G.

Wu N.H.

Nitsche A.

et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Zhou et al., 2020 Zhou P.

Yang X.-L.

Wang X.-G.

Hu B.

Zhang L.

Zhang W.

Si H.-R.

Zhu Y.

Li B.

Huang C.-L.

et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. The recent emergence of SARS-CoV-2 infection in China has led to major public health concerns. ACE2 has been reported to be the receptor for this novel CoV (). In this study, we determined the key region in SARS-CoV-2 that is responsible for the interaction with the receptor and solved the crystal structure of SARS-CoV-2-CTD in complex with hACE2.

Li et al., 2005 Li F.

Li W.

Farzan M.

Harrison S.C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Wu et al., 2009 Wu K.

Li W.

Peng G.

Li F. Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor. Li, 2015 Li F. Receptor recognition mechanisms of coronaviruses: a decade of structural studies. Considering the newly identified SARS-CoV-2, a total of seven human CoVs have been reported so far. Of these viruses, three (hCoV-NL63, SARS-CoV, and SARS-CoV-2) have been shown to utilize the hACE2 receptor for cell entry. The complex structures of hCoV-NL63 CTD and SARS-RBD bound to hACE2 have been reported previously (). Although hCoV-NL63 CTD and SARS-RBD are structurally distinct, the two viral ligands recognize and engage sterically overlapping sites in the receptor (). The complex structure of SARS-CoV-2-CTD together with hACE2 reveals that the majority of binding sites of SARS-CoV-2 in hACE2 also overlap the SARS-CoV binding site. The observations favor a scenario where these CoVs have evolved to recognize a “hotspot” region in hACE2 for receptor binding.

0AT1 (an amino acid transporter), as revealed by cryoelectron microscopy (cryo-EM) analysis ( Yan et al., 2020 Yan R.

Zhang Y.

Li Y.

Xia L.

Guo Y.

Zhou Q. Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2. 0AT1 bound to two SARS-CoV-2-CTDs, with each molecule bound to an hACE2 monomer, with a local resolution of 3.5 Å at the interface. Our crystal structure of SARS-CoV-2-CTD/hACE2 is well superimposed with the cryo-EM structure, with an RMSD of 1.019 Å over 722 pairs of Cα atoms. Notably, two cryo-EM structures of trimeric SARS-CoV-2 S proteins were also published recently, with the receptor binding region buried or exposed ( Walls et al., 2020 Walls A.C.

Park Y.J.

Tortorici M.A.

Wall A.

McGuire A.T.

Veesler D. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Wrapp et al., 2020 Wrapp D.

Wang N.

Corbett K.S.

Goldsmith J.A.

Hsieh C.L.

Abiona O.

Graham B.S.

McLellan J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Yuan et al., 2017 Yuan Y.

Cao D.

Zhang Y.

Ma J.

Qi J.

Wang Q.

Lu G.

Wu Y.

Yan J.

Shi Y.

et al. Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains. During the revision of our manuscript, the full-length hACE2 structure was reported to form a dimer in the presence of BAT1 (an amino acid transporter), as revealed by cryoelectron microscopy (cryo-EM) analysis (). They also reported the cryo-EM structure of dimeric hACE2-BAT1 bound to two SARS-CoV-2-CTDs, with each molecule bound to an hACE2 monomer, with a local resolution of 3.5 Å at the interface. Our crystal structure of SARS-CoV-2-CTD/hACE2 is well superimposed with the cryo-EM structure, with an RMSD of 1.019 Å over 722 pairs of Cα atoms. Notably, two cryo-EM structures of trimeric SARS-CoV-2 S proteins were also published recently, with the receptor binding region buried or exposed (), which is consistent with the structural features of MERS-CoV and SARS-CoV S proteins (). Further structure alignments show that the crystal structure of SARS-CoV-2-CTD in the complex also fits well with its counterparts in the cryo-EM structures, with RMSDs of 0.724 Å (exposed state) and 0.742 Å (buried states) related to PDB: 6VSB and 0.632 Å (exposed state) and 0.622 Å (buried state) related to PDB: 6VYB , respectively. These results indicate that the crystal structure of the complex is consistent with the respective cryo-EM structures and provide more detailed binding information.

Wrapp et al., 2020 Wrapp D.

Wang N.

Corbett K.S.

Goldsmith J.A.

Hsieh C.L.

Abiona O.

Graham B.S.

McLellan J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Considering the high sequence identity between SARS-CoV-2-CTD and SARS-RBD, atomic comparisons of the two viral ligands binding the same receptor were performed. Atomic details reveal more interactions in SARS-CoV-2-CTD/hACE2 than in SARS-RBD/hACE2, including more engaged residues, more vdw contacts, more H-bonds, as well as larger buried surface areas. Interestingly, the β1’/β2′ loop, which is the most variable region between SARS-CoV-2-CTD and SARS-RBD, confers more interactions to SARS-CoV-2-CTD/hACE2, including strong interactions, such as aromatic-aromatic interactions and ionic interactions, in contrast to the SARS-RBD β1’/β2′ loop. A recently published paper also indicates that the SARS-CoV-2 S protein binds hACE2 with higher affinity than the SARS-CoV S protein (), which was shown in this report as well.

Zhang et al., 2009 Zhang X.

Wang J.

Wen K.

Mou Z.

Zou L.

Che X.

Ni B.

Wu Y. Antibody binding site mapping of SARS-CoV spike protein receptor-binding domain by a combination of yeast surface display and phage peptide library screening. Wrapp et al., 2020 Wrapp D.

Wang N.

Corbett K.S.

Goldsmith J.A.

Hsieh C.L.

Abiona O.

Graham B.S.

McLellan J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Although SARS-CoV and SARS-CoV-2 share more than 70% sequence identity in the S protein, and both engage hACE2 via the CTD, we find that the two viruses CTDs are antigenically distinct. When using a panel of mAbs targeting SARS-CoV S1/CTD, none of the antibodies were able to recognize SARS-CoV-2 S. mAb1 and mAb2/mAb3 used in the above assay have been determined to bind to SARS-CoV S protein 330–350 and 380–399, respectively (). However, the binding sites for the other three mAbs (B30A38, A50A1A1, and C31A12), which were generated using SARS-CoV S1 as the immunogen, remain elusive. Consistently, a recently published paper also reported similar results showing that three SARS-RBD-directed mAbs, S230, m396, and 80R, were unable to bind to SARS-CoV-2 (). Furthermore, we also demonstrate that polyclonal antisera directed against SARS-RBD do not recognize the S protein of SARS-CoV-2. A comparison of the two viral ligands shows that they display divergent electrostatic potential, which likely results in differing immunogenicity despite both ligands showing a similar protein fold.