Structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections

Abstract

It is believed that a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), was passed from palm civets to humans and caused the epidemic of SARS in 2002 to 2003. The major species barriers between humans and civets for SARS-CoV infections are the specific interactions between a defined receptor-binding domain (RBD) on a viral spike protein and its host receptor, angiotensin-converting enzyme 2 (ACE2). In this study a chimeric ACE2 bearing the critical N-terminal helix from civet and the remaining peptidase domain from human was constructed, and it was shown that this construct has the same receptor activity as civet ACE2. In addition, crystal structures of the chimeric ACE2 complexed with RBDs from various human and civet SARS-CoV strains were determined. These structures, combined with a previously determined structure of human ACE2 complexed with the RBD from a human SARS-CoV strain, have revealed a structural basis for understanding the major species barriers between humans and civets for SARS-CoV infections. They show that the major species barriers are determined by interactions between four ACE2 residues (residues 31, 35, 38, and 353) and two RBD residues (residues 479 and 487), that early civet SARS-CoV isolates were prevented from infecting human cells due to imbalanced salt bridges at the hydrophobic virus/receptor interface, and that SARS-CoV has evolved to gain sustained infectivity for human cells by eliminating unfavorable free charges at the interface through stepwise mutations at positions 479 and 487. These results enhance our understanding of host adaptations and cross-species infections of SARS-CoV and other emerging animal viruses.

FIG. 1.

The SARS-CoV/receptor interface. (A) Alignment of residues on the SARS-CoV RBD at the interface that have undergone evolution. In red are two residues, residues 479 and 487, that determine the major species barriers between human and civet for SARS-CoV infections. Four prototypic viral strains are defined in the text. (B) Alignment of residues on the N-terminal helix of ACE2 that differ between human and civet. In red are residues that directly interact with SARS-CoV. (C) Crystal structure of the interface between hTor02 RBD and chimeric ACE2 bearing the N-terminal helix from civet and the remaining peptidase domain from human. The receptor-binding motif (RBM) on hTor02 RBD is in red, with side chains of the four residues that have undergone evolution (residues 472, 479, 480, and 487). The N-terminal helix from civet ACE2 is in green, with side chains of residues that differ between human and civet. The rest of peptidase domain from human ACE2 is in yellow. (D) An overall view of the crystal structure of human-civet chimeric ACE2 complexed with hTor02 RBD. The structure is rotated clockwise in depth compared with the structure in panel C. The illustrations were made using Povscript (4).

FIG. 2.

Solution RBD binding assays of human, civet, and chimeric ACE2. (A) Gel filtration chromatography on Superdex 200 of a mixture of human ACE2 and cSz02 RBD (top), a mixture of civet ACE2 and cSz02 RBD (middle), and a mixture of chimeric ACE2 and cSz02 RBD (bottom). The cSz02 RBD is in excess in each of the three mixtures. The dotted lines mark the relative elution volumes of the proteins. A leftward shift indicates binding. Peak a corresponds to unbound human ACE2. Peaks b and c correspond to cSz02 RBD-bound civet ACE2 and chimeric ACE2, respectively. (B) Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of peaks a, b, and c in panel A, confirming the protein components of each of the three peaks. The bands corresponding to RBD and ACE2 were confirmed by protein N-terminal sequencing (10). (C) Summary of receptor activities of human ACE2, civet ACE2, and chimeric ACE2. The latter two have the same receptor activity, which is different from that of human ACE2.

FIG. 3.

Structural basis for host adaptations of residue 479 on SARS-CoV RBD. (A) On the surface of unbound human ACE2 (28), Lys31 points into solution. (B) At the interface of human ACE2 and hTor02 (11), Lys31 on ACE2 folds back and forms a salt bridge with Glu35 on ACE2. Asn479 on RBD forms hydrophobic interactions with Tyr440 and Tyr442 on RBD. (C) At the interface of chimeric ACE2 and hTor02, Thr31 cannot form a salt bridge with Glu35 on civet ACE2. Consequently, Glu35 on civet ACE2 is unneutralized. (D) At the interface of chimeric ACE2 and cSz02, Lys479 on RBD forms a salt bridge with Glu35 on civet ACE2 and hydrophobic interactions with tyrosines. (E) At the interface of chimeric ACE2 and cGd05, Arg479 on RBD forms a strong bifurcated salt bridge with Glu35 on civet ACE2 and strong hydrophobic interactions with tyrosines. (F) Electron density map of the interface of chimeric ACE2 and cGd05, as part of a composite-omit map calculated from the refined model of chimeric ACE2 complexed with cGd05 RBD. The illustrations were made using Povscript (4).

FIG. 4.

Structural basis for host adaptations of residue 487 on SARS-CoV RBD. (A) On the surface of unbound human ACE2 (28), Lys353 points into solution. (B) At the interface of human ACE2 and hTor02 (11), Lys353 on ACE2 is embedded in a hydrophobic tunnel surrounded by Thr487 on hTor02 RBD and three tyrosines. Lys353 and Asp38 on ACE2 form a salt bridge, which requires support from RBD Thr487. (C) At the interface of chimeric ACE2 and hTor02, Glu38 and Lys353 on ACE2 form a bifurcated salt bridge, in the presence of RBD Thr487. (D) At the interface of chimeric ACE2 and cGd05, Glu38 and Lys353 on ACE2 form a strong bifurcated salt bridge, in the presence of RBD Ser487. (E) Electron density map of the interface of chimeric ACE2 and cGd05, as part of a composite-omit map calculated from the refined model of chimeric ACE2 complexed with cGd05 RBD. (F) Corey-Pauling-Koltun presentation of the hydrophobic tunnel surrounding ACE2 Lys353 at the interface of chimeric ACE2 and cGd05. The illustrations were made using Povscript (4).

FIG. 5.

Sequence alignment of SARS-CoV binding regions of ACE2s from 10 mammals. The GenBank accession numbers are AY623811 (human), AY881174 (civet), AB211998 (raccoon), NM_001039456 (cat), AB208708 (ferret), NM_001024502 (cattle), Q5RFN1 (orangutan), AY996037 (monkey), AY881244 (rat), and EF408740 (mouse). The alignment was generated by the ClusterW program. In red are residues that are critical to the major species barriers between hosts for SARS-CoV infections. In blue are residues that directly contact SARS-CoV. Asterisks indicate positions which have a single, fully conserved residue. Colons indicate positions which have strongly conserved residues. Periods indicate positions which have weakly conserved residues.