Molecular basis of hippopotamus ACE2 binding to SARS-CoV-2

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a wide range of hosts, including hippopotami, which are semi-aquatic mammals and phylogenetically closely related to Cetacea. In this study, we characterized the binding properties of hippopotamus angiotensin-converting enzyme 2 (hiACE2) to the spike (S) protein receptor binding domains (RBDs) of the SARS-CoV-2 prototype (PT) and variants of concern (VOCs). Furthermore, the cryo-electron microscopy (cryo-EM) structure of the SARS-CoV-2 PT S protein complexed with hiACE2 was resolved. Structural and mutational analyses revealed that L30 and F83, which are specific to hiACE2, played a crucial role in the hiACE2/SARS-CoV-2 RBD interaction. In addition, comparative and structural analysis of ACE2 orthologs suggested that the cetaceans may have the potential to be infected by SARS-CoV-2. These results provide crucial molecular insights into the susceptibility of hippopotami to SARS-CoV-2 and suggest the potential risk of SARS-CoV-2 VOCs spillover and the necessity for surveillance.

Importance: The hippopotami are the first semi-aquatic artiodactyl mammals wherein SARS-CoV-2 infection has been reported. Exploration of the invasion mechanism of SARS-CoV-2 will provide important information for the surveillance of SARS-CoV-2 in hippopotami, as well as other semi-aquatic mammals and cetaceans. Here, we found that hippopotamus ACE2 (hiACE2) could efficiently bind to the RBDs of the SARS-CoV-2 prototype (PT) and variants of concern (VOCs) and facilitate the transduction of SARS-CoV-2 PT and VOCs pseudoviruses into hiACE2-expressing cells. The cryo-EM structure of the SARS-CoV-2 PT S protein complexed with hiACE2 elucidated a few critical residues in the RBD/hiACE2 interface, especially L30 and F83 of hiACE2 which are unique to hiACE2 and contributed to the decreased binding affinity to PT RBD compared to human ACE2. Our work provides insight into cross-species transmission and highlights the necessity for monitoring host jumps and spillover events on SARS-CoV-2 in semi-aquatic/aquatic mammals.

Keywords: ACE2; SARS-CoV-2; cross-species recognition; cryo-EM structure; hippopotamus; receptor binding domain (RBD); spike (S).

Fig 1

Characterization of binding between SARS-CoV-2 RBDs and hiACE2, and the infectivity of pseudotyped SARS-CoV-2 in BHK-21 cells expressing hiACE2. (A) Flow cytometry assay of SARS-CoV-2 RBDs binding to hiACE2-expressing BHK-21 cells. (B) The percentage of SARS-CoV-2 RBD-positive BHK-21 cells expressing hiACE2. The data represent the results of three replicates. (C) SPR characterization of RBDs from SARS-CoV-2 PT and its variants interacting with hiACE2. The raw and fitted curves are displayed as dotted and red solid lines, respectively. The dissociation constant (K_D) calculated from the results of three independent repeated experiments is presented as the mean ± standard deviation. (D) Transduction of the pseudotyped SARS-CoV-2 PT and VOCs in BHK-21 cells expressing hiACE2. The data represent the results of six replicates.

Fig 2

Overall architectures of SARS-CoV-2 PT RBD binding to hiACE2 and hACE2 complexes. (A-B) The overall complex structures of SARS-CoV-2 PT RBD binding to hiACE2 (A) and hACE2 (B). The boxes indicate the interaction patches. The H-bonds networks of Patch 1 and Patch 2 are shown as red dotted lines. A cartoon representation of the complex structures is shown, and residues participating in H-bond formation are shown as sticks.

Fig 3

The binding interfaces and interaction networks of the SARS -CoV-2 PT RBD with hiACE2 and hACE2. (A-B) The binding interfaces of hiACE2/SARS-CoV-2 PT RBD complex (A) and hACE2/SARS-CoV-2 PT RBD (B) complex. (C) The interaction networks of SARS-CoV-2 PT RBD with hiACE2 or hACE2. Black lines indicate vdW contacts (within 4.5 Å), and red lines represent H-bonds or salt bridges (within 3.5 Å).

Fig 4

Structural comparison of key residues in the molecular interaction of hiACE2/SARS-CoV-2 PT RBD and hACE2/SARS-CoV-2 PT RBD. (A and B) The structural details of comparison of hACE2/SARS-CoV-2 PT-RBD with hiACE2/SARS-CoV-2 PT RBD around the sites 30 (A) and 83 (B) of ACE2. The residues involved in the interaction are represented as sticks. The H-bonds between ACE2 and the RBD residues are indicated by red dotted lines. The SARS-CoV-2 PT RBD, hiACE2, and hACE2 are shown in cyan, green, and yellow colors, respectively. (C) SPR analysis of the binding affinity of SARS-CoV-2 PT RBD for hiACE2, hACE2, and mutated hiACE2. The raw and fitted curves are displayed in dotted and red solid lines, respectively. The dissociation constant (K_D) calculated from the results of three independent repeated experiments is presented as the mean ± standard deviation. (D) Sequence alignment of residues in ACE2s from different species in contact with the RBD. The conserved residues in ACE2 orthologs are shown as black letters. The residue substitutions in ACE2 from other species relative to hACE2 are shown in red letters, and the L30 of hi ACE2 is marked with blue letters to highlight its unique hydrophobicity. The orange background indicates species that have been naturally or experimentally infected with SARS-CoV-2, while the blue background indicates species with a high-risk and resolved ACE2-RBD complex structure.

Fig 5

Comparative analysis of key residues in ACE2 between hippopotami and cetaceans. (A) Sequence alignment of residues in ACE2s from whippomorpha species in contact with the RBD. The conserved residues in ACE2 orthologs are shown as black letters. The residue substitutions in ACE2 from other species relative to hiACE2 are shown as red letters, (B) The structural details of comparison of hiACE2/SARS-CoV-2 PT RBD (wheat) with MW-ACE2/SARS-CoV-2 PT RBD (cyan) around the sites 30, 35, 42, 82, and 83 of ACE2. Residues involved in the interaction are represented as sticks. The H-bonds between ACE2 and the RBD residues are indicated by red dotted lines.