SARS2 ja ACE2

https://www.biorxiv.org/content/10.1101/2020.02.17.951848v
 Abstract

Angiotensin-converting enzyme 2 (ACE2) is the surface receptor for SARS coronavirus (SARS-CoV), directly interacting with the spike glycoprotein (S protein). ACE2 is also suggested to be the receptor for the new coronavirus (2019-nCoV), which is causing a serious epidemic in China manifested with severe respiratory syndrome. B⁰AT1 (SLC6A19) is a neutral amino acid transporter whose surface expression in intestinal cells requires ACE2. Here we present the 2.9 Å resolution cryo-EM structure of full-length human ACE2 in complex with B⁰AT1. The complex, assembled as a dimer of ACE2-B⁰AT1 heterodimers, exhibits open and closed conformations due to the shifts of the peptidase domains (PDs) of ACE2. A newly resolved Collectrin-like domain (CLD) on ACE2 mediates homo-dimerization. Structural modelling suggests that the ACE2-B⁰AT1 complex can bind two S proteins simultaneously, providing important clues to the molecular basis for coronavirus recognition and infection.

Introduction

The 2019-nCoV is a type of positive-stranded RNA viruses that causes severe respiratory syndrome in human. The resulting outbreak of the coronavirus disease 2019 (COVID-19) has emerged as a severe epidemic, claiming more than one hundred lives everyday (1, 2). The genome of 2019-nCoV shares ~ 80% identity with that of SARS-CoV and ~ 96% identical to a bat coronavirus (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 maturation, the trimeric S protein is cleaved into S1 and S2 subunits (4, 5). S1 contains the receptor binding domain (RBD), which directly binds to ACE2 (6), and S2 is responsible for membrane fusion. When S1 binds to the host receptor ACE2, another cleavage site on S2 is exposed and cut by host proteases, a process that is critical for successful viral infection (5, 7, 8). It has been shown that the S protein of 2019-nCoV, same as SARS-CoV, may exploit ACE2 for host infection (2, 9–11). In particular, a most recent bioRxiv preprint suggests that the affinity between ACE2 and the RBD of 2019-nCoV is 10-20 times higher than that with the RBD of SARS-CoV (12).

Although ACE2 is known to the public because it is hijacked by coronaviruses, the primary physiological role of ACE2, as its name indicates, is to facilitate maturation of angiotensin, a peptide hormone that controls vasoconstriction and blood pressure. ACE2 is a type I membrane protein expressed in lung, heart, kidney and intestine (13–15). The expression level of ACE2 is associated with cardiovascular diseases (16–18). The full-length ACE2 consists of an N-terminal peptidase domain (PD) and a C-terminal Collectrin-like domain (CLD) that ends with a single transmembrane helix and a ~ 40-residue intracellular segment (13, 19). The PD of ACE2 cleaves angiotensin (Ang) I to produce Ang-(1–9), which is then processed by other enzymes to become Ang-(1–7). ACE2 can also directly process angiotensin II to result in Ang-(1–7) (13, 20).

The PD of ACE2 also provides the direct binding site for the S proteins of coronaviruses. Crystal structure of the claw-like ACE2-PD (21) and structures of the complex between PD and S protein of SARS-CoV have revealed the molecular details of the interaction between the RBD of S protein and PD of ACE2 (6, 22, 23). Apart from PD, the rest of ACE2 is structurally unavailable. The single-TM of ACE2 represents a major challenge for structural determination of the full-length protein.

Recent studies show that ACE2 moonlights as the chaperone for the membrane trafficking of an amino acid transporter, B⁰AT1, also known as SLC6A19 (24). B⁰AT1 mediates uptake of neutral amino acids into intestinal cells in a sodium dependent manner. Its deficiency may cause the Hartnup disorder, an inherited disease with pellagra, cerebellar ataxia and psychosis (25–27). Structures of SLC6 members of dDAT (Drosophila Dopamine transporter) and human SERT (Serotonin transporter, SLC6A4) have been reported (28, 29). It is unclear how ACE2 interacts with B⁰AT1 and facilitates its membrane trafficking. Our structure of LAT1-4F2hc shows that the cargo and the chaperon interact through both extracellular and transmembrane domains (30). We reasoned that the structure of the full-length ACE2 may be revealed in the presence of B⁰AT1.

Here we report the cryo-EM structure of the full-length human ACE2-B⁰AT1 complex at an overall resolution of 2.9 Å. The complex exists as a dimer of heterodimers. Two conformations of the PD in the ACE2 homodimer, open and close, were extracted from the same dataset. Docking of the structure of the complex between ACE2-PD and the SARS-CoV S protein suggests that two S protein trimers can simultaneously bind to an ACE2 homodimer.

Structural determination of ACE2-B⁰AT1 complex

The full-length human ACE2 and B⁰AT1, with Strep and FLAG tags on their respective N-terminus, were co-expressed in HEK293F cells and purified through tandem affinity resin and size exclusion chromatography. The complex was eluted in a single mono-disperse peak, indicating high homogeneity (Fig. 1A). Details of cryo-sample preparation, data acquisition, and structural determination are illustrated in Materials and Methods. Eventually, out of 418,140 selected particles, a 3D reconstruction was obtained at an overall resolution of 2.9 Å. An organization of dimer of the ACE2-B⁰AT1 heterodimers was immediately revealed (Fig. 1B). After application of focused refinement and C2 symmetry expansion, the resolution of the extracellular domains was improved to 2.7 Å, while the TM domain remains the same (Figs. 1B, Supplementary Figures 1-3, Table 1).

The high resolution supported reliable model building. For ACE2, side chains can be assigned to residues 19-768 that contain the PD (residues 19-615) and the newly revealed CLD consisting of a small extracellular domain, a long linker, and the single TM (Fig. 1C). The small domain, sharing a ferredoxin-like fold, will be referred to as the Neck domain because it connects the PD and the TM (Fig. 1C, Supplementary Figure 4). The homo-dimerization is entirely mediated by ACE2, which is sandwiched by B⁰AT1. Both the PD and Neck domain contribute to dimerization, while each B⁰AT1 interacts with the Neck and TM in the adjacent ACE2 (Fig. 1C). The extracellular region is highly glycosylated, with 7 and 5 glycosylation sites on ACE2 and B⁰AT1, respectively.

During classification, another subset with 143,857 particles was processed to an overall resolution of 4.5 Å. It is apparent that while the Neck domain still dimerizes, the PDs are separated from each other in this reconstruction (Fig. 1D, Supplementary Figure 1H-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 only, with the rest of the complex nearly unchanged (Supplementary Movie 1).

Homodimer interface of ACE2

Dimerization of ACE2 is mainly mediated by the Neck domain, with PD contributing a minor interface (Fig. 2A). To facilitate structural illustrations, we will refer to the two ACE2 protomers A and B, with residues in protomer B labeled with’. Extensive polar interactions are mapped to the interface between N2 and N4 helices on the Neck domain (Fig. 2B). Arg652 and Arg710 in ACE2-A form cation-π interaction with Tyr641’ and Tyr633’ from ACE2-B, respectively. Meanwhile, Arg652 and Arg710 are respectively hydrogen-bonded (H-bond) to Asn638’ and Glu639’, which simultaneously interact with Gln653, in addition to Asn636’. Ser709 and Asp713 from ACE2-A are H-bonded to Arg716’. This extensive network of polar interactions indicates a stable dimer formation.

The PD dimer interface appears much weaker with only one pair of interactions between Gln139 and Gln175’ (Fig. 2C). Note that Gln139 is positioned on a loop which is stabilized by a disulfide bond between Cys133 and Cys141 as well as multiple intro-loop polar interactions (Fig. 2C). The weak interaction is consistent with the presence of 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, Supplementary Movie 1).

Two trimeric S proteins can bind to an ACE2 dimer simultaneously

The available structures for the complex between the SARS-CoV S protein and ACE2 are limited to the isolated PD only. We next examined whether a dimeric ACE2 can simultaneously accommodate two S protein trimers. When the S protein-PD complex (PDB code: 6ACJ) is superimposed with the PD in the closed or open states of the ACE2-B⁰AT1 complex, the PD domains can be aligned with root mean squared deviation of ~ 1.9 Å over ~ 550 pairs of C_α atoms with the closed conformation or of ~ 4.9 Å over ~ 590 pairs of C_α atoms with the open conformation (Fig. 3A, B, Supplementary Figure 5). In both states, S protein is positioned on the outer side of ACE2 dimer. Therefore, a dimeric ACE2 can accommodate two S protein trimers, simultaneously. This observation immediately implies potential clustering between 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 receptor-mediated endocytosis.

Because our proteins were expressed in mammalian cells, whereas the published ACE2-PD was obtained from baculovirus-infected insect cells, more glycosylation sites, six vs two, were observed on the surface of PD (Fig. 3C). The significance of glycosylation in the recognition between viruses and receptors has drawn increasing attention. Among the observed glycosylation sites on PD, three are in the vicinity of the RBD binding site (Fig. 3D). Two, Asn90 and Asn322, were observed previously (6), and one, Asn53, is revealed in this study. It has been shown that chloroquine, which can interfere with the terminal glycosylation of ACE2 (31), inhibits SARS-CoV infection. It remains to be shown whether these sugar moieties directly participate in S protein binding.

The interfaces between ACE2 and B⁰AT1

As the PD and Neck domain are connected to the TM though an elongated linker, it is likely that the extended conformation is stabilized by B⁰AT1. Indeed, a close look at the heterodimer shows extensive interface along one side of the Neck domain and TM of ACE2 with an extended TM7 and TM4 of B⁰AT1 (Fig. 4A), reminiscent to the interactions between 4F2hc and LAT1 (30, 32).