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Crystal structure of bovine coronavirus spike protein lectin domain - PubMed

  • ️Sun Jan 01 2012

Crystal structure of bovine coronavirus spike protein lectin domain

Guiqing Peng et al. J Biol Chem. 2012.

Abstract

The spike protein N-terminal domains (NTDs) of bovine coronavirus (BCoV) and mouse hepatitis coronavirus (MHV) recognize sugar and protein receptors, respectively, despite their significant sequence homology. We recently determined the crystal structure of MHV NTD complexed with its protein receptor murine carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which surprisingly revealed a human galectin (galactose-binding lectin) fold in MHV NTD. Here, we have determined at 1.55 Å resolution the crystal structure of BCoV NTD, which also has the human galectin fold. Using mutagenesis, we have located the sugar-binding site in BCoV NTD, which overlaps with the galactose-binding site in human galectins. Using a glycan array screen, we have identified 5-N-acetyl-9-O-acetylneuraminic acid as the preferred sugar substrate for BCoV NTD. Subtle structural differences between BCoV and MHV NTDs, primarily involving different conformations of receptor-binding loops, explain why BCoV NTD does not bind CEACAM1 and why MHV NTD does not bind sugar. These results suggest a successful viral evolution strategy in which coronaviruses stole a galectin from hosts, incorporated it into their spike protein, and evolved it into viral receptor-binding domains with altered sugar specificity in contemporary BCoV or novel protein specificity in contemporary MHV.

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Figures

FIGURE 1.
FIGURE 1.

Crystal structure of BCoV NTD. A, overall structure of BCoV NTD. Two β-sheets of NTD core are colored green and magenta, respectively, and other parts of NTD are colored cyan. N*, N terminus; C*, C terminus. The β-sandwich core structure is indicated as “core.” The two potential sugar-binding pockets above and underneath the core structure are indicated as top and bottom, respectively. B, 2Fo − Fc electron density of a portion of BCoV NTD at 1.5σ. This region includes three of the critical sugar-binding residues. C, secondary structures of BCoV NTD and sequence alignment of BCoV, HCoV-OC43, and MHV NTDs. β-Strands are shown as arrows, and α-helices are shown as cylinders. The sequences are colored the same way as the corresponding secondary structures in A. In MHV NTD, two highlighted regions, one covering β2′ and part of β3 and the other at the C terminus, are disordered (22). Also in MHV NTD, the four highlighted and red-colored regions are CEACAM1-binding RBMs (RBM1–4 from N to C terminus). In BCoV and HCoV-OC43 NTDs, the four highlighted and brown-colored residues between β11 and β13 are critical sugar-binding residues. In all three NTDs, the highlighted region covering part of β10 and loop 10–11 varies significantly in length. BCoV strain, Mebus; HCoV-OC43 strain, ATCC VR759; MHV strain, A59. Asterisks indicate positions that have fully conserved residues. Colons indicate positions that have strongly conserved residues. Periods indicate positions that have weakly conserved residues.

FIGURE 2.
FIGURE 2.

Stereo image of the superimposed structures of BCoV and MHV NTDs. BCoV NTD is colored blue, and MHV NTD is colored green. Two of the mCEACAM1a-binding loops in MHV NTD are colored red and labeled as receptor-binding motifs 1 and 4 (RBM1 and RBM4). Sugar-binding residues in BCoV NTD are colored brown and shown in stick-and-ball presentation. Bidirectional arrows indicate different conformations of the receptor-binding loops in the two NTDs. One-directional arrows indicate the location of mCEACAM1a that binds MHV NTD.

FIGURE 3.
FIGURE 3.

Interactions between coronavirus NTDs and mammalian CEACAM1 proteins. A, relative receptor-binding activities of coronavirus NTDs by ELISA. Measured were relative binding activities between coronavirus NTDs and mammalian CEACAM1 proteins that had been coated on 96-well Maxisorp plates. CEACAM1-binding NTDs were detected using antibodies against their C-terminal His6 tags. As a comparison, binding activities between coronavirus NTDs and sugar moieties on mucin-coated plates were also shown. PBS buffer was used as a negative control. All of the binding activities have been calibrated against the binding activity between MHV NTD and mCEACAM1a. B, receptor-binding affinities of coronavirus NTDs by surface plasmon resonance using Biacore. Mammalian CEACAM1 proteins were immobilized on Biacore chips, and coronavirus NTDs were flown through. N.A. indicates that the binding affinity is too low to be reliably measured. As a comparison, binding affinities between coronavirus NTDs and sugar moieties on mucin-immobilized sensor chips were also shown. mCEACAM1, murine CEACAM1; bCEACAM1, bovine CEACAM1; hCEACAM1, human CEACAM1.

FIGURE 4.
FIGURE 4.

Interactions between BCoV NTD and sugar. A, relative binding activities between BCoV NTD and sugar moieties on mucin-coated plates by ELISA. Sugar-binding BCoV NTD was detected using antibodies against its C-terminal His6 tag. Sugar-binding activities of both wild-type and mutant BCoV NTDs were measured. All of the sugar-binding activities have been calibrated against the sugar-binding activity of wild-type BCoV NTD. B, binding affinity between BCoV NTD and sugar moieties on mucin by surface plasmon resonance using Biacore. Mucin was immobilized on Biacore chips, and BCoV NTD was flown through. C, distribution of mutated residues in the pocket above the β-sandwich core. Critical sugar-binding residues are colored brown, and non-critical residues are colored yellow. D, distribution of mutated residues in the pocket underneath the β-sandwich core. Surface presentations of the pockets were shown as semi-transparent white surfaces. N.A., not available.

FIGURE 5.
FIGURE 5.

Glycan screen array to identify substrate sugar type for BCoV NTD. See

supplemental Table S1

for glycans used in the experiment. Among these glycans, 5-N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) shows the highest affinity for BCoV NTD. RFU, relative fluorescence unit.

FIGURE 6.
FIGURE 6.

Comparison of sugar-binding sites in BCoV NTD and human galectins. A, sugar-binding site in BCoV NTD. Black circle indicates the Neu5,9Ac2-binding site in BCoV NTD, as identified by mutagenesis studies. Critical sugar-binding residues are colored brown, and non-critical residues are colored yellow. B, galactose-binding site in human galectin 3 (Protein Data Bank code 1A3K). Galactose is colored gray, and critical galactose-binding residues are colored brown.

FIGURE 7.
FIGURE 7.

Proposed origin and evolution of coronavirus spike protein lectin domain. Orange arrows indicate the locations of CEACAM1 or sugar that binds coronavirus NTDs. Question marks indicate the postulated structures of hypothetical evolutionary intermediates.

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