Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

How SARS-CoV-2 binds to human cells Scientists are racing to learn the secrets of severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2), which is the cause of the pandemic disease COVID-19. The first step in viral entry is the binding of the viral trimeric spike protein to the human receptor angiotensin-converting enzyme 2 (ACE2). Yan et al. present the structure of human ACE2 in complex with a membrane protein that it chaperones, B0AT1. In the context of this complex, ACE2 is a dimer. A further structure shows how the receptor binding domain of SARS-CoV-2 interacts with ACE2 and suggests that it is possible that two trimeric spike proteins bind to an ACE2 dimer. The structures provide a basis for the development of therapeutics targeting this crucial interaction. Science, this issue p. 1444

S evere acute respiratory syndromecoronavirus 2 (SARS-CoV-2) is a positivestrand RNA virus that causes severe respiratory syndrome in humans. The resulting outbreak of coronavirus disease 2019 (COVID-19) has emerged as a severe epidemic, claiming more than 2000 lives worldwide between December 2019 and February 2020 (1,2). The genome of SARS-CoV-2 shares about 80% identity with that of SARS-CoV and is about 96% identical to the bat coronavirus BatCoV RaTG13 (2).
In the case of SARS-CoV, the spike glycoprotein (S protein) on the virion surface mediates receptor recognition and membrane fusion (3,4). During viral infection, the trimeric S protein is cleaved into S1 and S2 subunits and S1 subunits are released in the transition to the postfusion conformation (4)(5)(6)(7). S1 contains the receptor binding domain (RBD), which directly binds to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2) (8), whereas S2 is responsible for membrane fusion. When S1 binds to the host receptor ACE2, another cleavage site on S2 is exposed and is cleaved by host proteases, a process that is critical for viral infection (5,9,10). The S protein of SARS-CoV-2 may also exploit ACE2 for host infection (2,(11)(12)(13). A recent publication reported the structure of the S protein of SARS-CoV-2 and showed that the ectodomain of the SARS-CoV-2 S protein binds to the PD of ACE2 with a dissociation constant (K d ) of~15 nM (14).
Although ACE2 is hijacked by some coronaviruses, its primary physiological role is in the maturation of angiotensin (Ang), a peptide hormone that controls vasoconstriction and blood pressure. ACE2 is a type I membrane protein expressed in lungs, heart, kidneys, and intestine (15)(16)(17). Decreased expression of ACE2 is associated with cardiovascular diseases (18)(19)(20). Full-length ACE2 consists of an Nterminal PD and a C-terminal collectrin-like domain (CLD) that ends with a single transmembrane helix and a~40-residue intracellular segment (15,21). The PD of ACE2 cleaves Ang I to produce Ang-(1-9), which is then processed by other enzymes to become Ang-(1-7). ACE2 can also directly process Ang II to give Ang-(1-7) (15,22).
Structures of the claw-like ACE2-PD alone and in complex with the RBD or the S protein of SARS-CoV have revealed the molecular details of the interaction between the RBD of the S protein and PD of ACE2 (7,8,23,24). Structural information on ACE2 is limited to the PD domain. The single transmembrane (TM) helix of ACE2 makes it challenging to determine the structure of the full-length protein.
ACE2 also functions as the chaperone for membrane trafficking of the amino acid transporter B 0 AT1, also known as SLC6A19 (25), which mediates uptake of neutral amino acids into intestinal cells in a sodium-dependent manner. Mutations in B 0 AT1 may cause Hartnup disorder, an inherited disease with symptoms such as pellagra, cerebellar ataxia, and psychosis (26)(27)(28). Structures have been determined for the SLC6 family members dDAT (Drosophila dopamine transporter) and human SERT (serotonin transporter, SLC6A4) (29,30). It is unclear how ACE2 interacts with B 0 AT1. The membrane trafficking mechanism for ACE2 and B 0 AT1 is similar to that of the LAT1-4F2hc complex, a large neutral-amino acid transporter complex that requires 4F2hc for its plasma membrane localization (31). Our structure of LAT1-4F2hc shows that the cargo LAT1 and chaperone 4F2hc interact through both extracellular and transmembrane domains (32). We reasoned that the structure of full-length ACE2 may be revealed in the presence of B 0 AT1.
Here, we report cryo-electron microscopy (cryo-EM) structures of the full-length human ACE2-B 0 AT1 complex at an overall resolution of 2.9 Å and a complex between the RBD of SARS-CoV-2 and the ACE2-B 0 AT1 complex, also with an overall resolution of 2.9 Å and with 3.5-Å local resolution at the ACE2-RBD interface. The ACE2-B 0 AT1 complex exists as a dimer of heterodimers. Structural alignment of the RBD-ACE2-B 0 AT1 ternary complex with the S protein of SARS-CoV-2 suggests that two S protein trimers can simultaneously bind to an ACE2 homodimer.
Structural determination of the ACE2-B 0 AT1 complex Full-length human ACE2 and B 0 AT1, with Strep and FLAG tags on their respective N termini, were coexpressed in human embryonic kidney (HEK) 293F cells and purified through tandem affinity resin and size exclusion chromatography. The complex was eluted in a single monodisperse peak, indicating high homogeneity (Fig. 1A). Details of cryo-sample preparation, data acquisition, and structural determination are given in the materials and methods section of the supplementary materials. A threedimensional (3D) reconstruction was obtained at an overall resolution of 2.9 Å from 418,140 selected particles. This immediately revealed the dimer of heterodimers' architecture ( Fig.  1B). After applying focused refinement and C2 symmetry expansion, the resolution of the extracellular domains improved to 2.7 Å, whereas the TM domain remained at 2.9-Å resolution ( Fig. 1B, figs. S1 to S3, and table S1).
The high resolution supported reliable model building. For ACE2, side chains could be assigned to residues 19 to 768, which contain the PD (residues 19 to 615) and the CLD (residues 616 to 768), which consists of a small extracellular domain, a long linker, and the single TM helix (Fig. 1C). Between the PD and TM helix is a ferredoxin-like fold domain; we refer to this as the neck domain (residues 616 to 726) ( Fig. 1C and fig. S4). Homodimerization is entirely mediated by ACE2, which is sandwiched by B 0 AT1. Both the PD and neck domains contribute to dimerization, whereas each B 0 AT1 interacts with the neck and TM helix in the adjacent ACE2 (Fig. 1C). The extracellular region is highly glycosylated, with seven and five glycosylation sites on each ACE2 and B 0 AT1 monomer, respectively.
During classification, another subset with 143,857 particles was processed to an overall resolution of 4.5 Å. Whereas the neck domain still dimerizes, the PDs are separated from each other in this reconstruction ( Fig.  1D and fig. S1, H to K). We therefore define the two classes as the open and closed conformations. Structural comparison shows that the conformational changes are achieved through rotation of the PD domains, with the rest of the complex left nearly unchanged (movie S1).

Homodimer interface of ACE2
Dimerization of ACE2 is mainly mediated by the neck domain, with the PD contributing a minor interface (Fig. 2A). The two ACE2 protomers are hereafter referred to as A and B, with residues in protomer B followed by a prime symbol. Extensive polar interactions are mapped to the interface between the second (residues 636 to 658) and fourth (residues 708 to 717) helices of the neck domain (Fig. 2B). Arg 652 and Arg 710 in ACE2-A form cation-p interactions with Tyr 641′ and Tyr 633′ in ACE2-B. Meanwhile, Arg 652 and Arg 710 are respectively hydrogen-bonded (H-bonded) to Asn 638′ and Glu 639′ , which also interact with Gln 653 , as does Asn 636′ . Ser 709 and Asp 713 from ACE2-A are H-bonded to Arg 716′ . This extensive network of polar interactions indicates stable dimer formation.
The PD dimer interface appears much weaker, with only one pair of interactions between Gln 139 and Gln 175′ (Fig. 2C). Gln 139 is in a loop that is stabilized by a disulfide bond between Cys 133 and Cys 141 as well as multiple intraloop polar interactions (Fig. 2C). The weak interaction is consistent with the ability to transition to the open conformation, in which the interface between the neck domains remains the same while the PDs are separated from each other by~25 Å ( Fig. 2D and movie S1).
Overall structure of the RBD-ACE2-B 0 AT1 complex To gain insight into the interaction between ACE2 and SARS-CoV-2, we purchased 0.2 mg of recombinantly expressed and purified RBD-mFc of SARS-CoV-2 (for simplicity, hereafter referred to as RBD; mFc, mouse Fc tag) from Sino Biological Inc., mixed it with our purified ACE2-B 0 AT1 complex at a stoichiometric ratio of~1.1 to 1, and proceeded with cryogrid preparation and imaging. Finally, a 3D EM reconstruction of the ternary complex was obtained.
In contrast to the ACE2-B 0 AT1 complexwhich has two conformations, open and closedonly the closed state of ACE2 was observed in the dataset for the RBD-ACE2-B 0 AT1 ternary complex. The structure of the ternary complex was determined to an overall resolution of 2.9 Å from 527,017 selected particles. However, the resolution for the ACE2-B 0 AT1 complex was substantially higher than that for the RBDs, which are at the periphery of the complex (Fig. 3A). To improve the local resolution, focused refinement was applied; this allowed us to reach a resolution of 3.5 Å for the RBD, supporting reliable modeling and analysis of the interface (Fig. 3, figs. S5 to S7, and table S1).

Interface between the RBD and ACE2
As expected, each PD accommodates one RBD (Fig. 3B). The overall interface is similar to that between SARS-CoV and ACE2 (7,8), mediated mainly through polar interactions (Fig. 4A). An extended loop region of the RBD spans the arch-shaped a1 helix of the ACE2-PD like a bridge. The a2 helix and a loop that connects the b3 and b4 antiparallel strands, referred 2 of 5 to as loop 3-4, of the PD also make limited contributions to the coordination of the RBD.
The contact can be divided into three clusters. The two ends of the bridge interact with the N and C termini of the a1 helix as well as small areas on the a2 helix and loop 3-4. The middle segment of a1 reinforces the interaction by engaging two polar residues (Fig.  4A). At the N terminus of a1, Gln 498 , Thr 500 , and Asn 501 of the RBD form a network of H-bonds with Tyr 41 , Gln 42 , Lys 353 , and Arg 357 from ACE2 (Fig. 4B). In the middle of the bridge, Lys 417 and Tyr 453 of the RBD interact with Asp 30 and His 34 of ACE2, respectively (Fig.  4C). At the C terminus of a1, Gln 474 of the RBD is H-bonded to Gln 24 of ACE2, whereas Phe 486 of the RBD interacts with Met 82 of ACE2 through van der Waals forces (Fig. 4D).

Comparing the SARS-CoV-2 and SARS-CoV interfaces with ACE2
Superimposition of the RBD in the complex of SARS-CoV (SARS-CoV-RBD) and ACE2-PD [Protein Data Bank (PDB) 2AJF] with the RBD in our ternary complex shows that the SARS-CoV-2 RBD (SARS-CoV-2-RBD) is similar to SARS-CoV-RBD with a root mean square deviation (RMSD) of 0.68 Å over 139 pairs of Ca atoms (Fig. 5A) (8). Despite the overall similarity, a number of sequence variations and conformational deviations are found in their respective interfaces with ACE2 ( Fig. 5 and fig. S8   The inset shows the focused refined map of RBD. The color scheme is the same as that in Fig.  1B, with the addition of red and gold, which represent RBD protomers. (B) Overall structure of the RBD-ACE2-B 0 AT1 complex. The color scheme is the same as that in Fig. 1C. The glycosylation moieties are shown as sticks.

Discussion
Although ACE2 is a chaperone for B 0 AT1, our focus is on ACE2 in this study. With the stabilization by B 0 AT1, we elucidated the structure of full-length ACE2. B 0 AT1 is not involved in dimerization, suggesting that ACE2 may be a homodimer even in the absence of B 0 AT1. Further examination suggests that a dimeric ACE2 can accommodate two S protein trimers, each through a monomer of ACE2 (fig. S9). The trimeric structure of the S protein of SARS-CoV-2 was recently reported, with one RBD in an up conformation and two in down conformations (PDB 6VSB) (14). The PD clashes with the rest of the S protein when the ternary complex is aligned to the RBD of the down conformation. There is no clash when the complex is superimposed on RDB in the up conformation, with a RMSD of 0.98 Å over 126 pairs of C a atoms, confirming that an up conformation of RDB is required to bind to the receptor ( fig. S9) (14).
Cleavage of the S protein of SARS-CoV is facilitated by cathepsin L in endosomes, indicating a mechanism of receptor-mediated endocytosis (10). Further characterization is required to examine the interactions between ACE2 and the viral particle as well as the effect of cofactors on this process (25,33). It remains to be investigated whether there is clustering between the dimeric ACE2 and trimeric S proteins, which may be important for invagination of the membrane and endocytosis of the viral particle, a process similar to other types of receptormediated endocytosis.
Cleavage of the C-terminal segment, especially residues 697 to 716 ( fig. S4), of ACE2 by proteases, such as transmembrane protease serine 2 (TMPRSS2), enhances the S proteindriven viral entry (34,35). Residues 697 to 716 form the third and fourth helices in the neck domain and map to the dimeric interface of ACE2. The presence of B 0 AT1 may block the access of TMPRSS2 to the cutting site on ACE2. The expression distribution of ACE2 is broader than that of B 0 AT1. In addition to kidneys and intestine, where B 0 AT1 is mainly expressed, ACE2 is also expressed in lungs and heart (27). It remains to be tested whether B 0 AT1 can suppress SARS-CoV-2 infection by blocking ACE2 cleavage. Enteric infections have been reported for SARS-CoV, and possibly also for SARS-CoV-2 (36,37). B 0 AT1 has also been shown to interact with another coronavirus receptor, aminopeptidase N (APN or CD13) (38). These findings suggest that B 0 AT1 may play a regulatory role for the enteric infections of some coronaviruses.
Comparing   (Fig. 5D). However, replacement of Arg 426 with Asn 439 appears to weaken the interaction by eliminating one important salt bridge with Asp 329 on ACE2 (Fig. 5B). Our structural work reveals the high-resolution structure of full-length ACE2 in a dimeric assembly. Docking the S protein trimer onto the structure of the ACE2 dimer with the RBD of the S protein bound suggests simultaneous binding of two S protein trimers to an ACE2 dimer. Structure-based rational design of binders with enhanced affinities to either ACE2 or the S protein of the coronaviruses may facilitate development of decoy ligands or neutralizing antibodies for suppression of viral infection. permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.