The SARS coronavirus S glycoprotein receptor binding domain: fine mapping and functional characterization - PubMed (original) (raw)

The SARS coronavirus S glycoprotein receptor binding domain: fine mapping and functional characterization

Samitabh Chakraborti et al. Virol J. 2005.

Abstract

The entry of the SARS coronavirus (SCV) into cells is initiated by binding of its spike envelope glycoprotein (S) to a receptor, ACE2. We and others identified the receptor-binding domain (RBD) by using S fragments of various lengths but all including the amino acid residue 318 and two other potential glycosylation sites. To further characterize the role of glycosylation and identify residues important for its function as an interacting partner of ACE2, we have cloned, expressed and characterized various soluble fragments of S containing RBD, and mutated all potential glycosylation sites and 32 other residues. The shortest of these fragments still able to bind the receptor ACE2 did not include residue 318 (which is a potential glycosylation site), but started at residue 319, and has only two potential glycosylation sites (residues 330 and 357). Mutation of each of these sites to either alanine or glutamine, as well as mutation of residue 318 to alanine in longer fragments resulted in the same decrease of molecular weight (by approximately 3 kDa) suggesting that all glycosylation sites are functional. Simultaneous mutation of all glycosylation sites resulted in lack of expression suggesting that at least one glycosylation site (any of the three) is required for expression. Glycosylation did not affect binding to ACE2. Alanine scanning mutagenesis of the fragment S319-518 resulted in the identification of ten residues (K390, R426, D429, T431, I455, N473, F483, Q492, Y494, R495) that significantly reduced binding to ACE2, and one residue (D393) that appears to increase binding. Mutation of residue T431 reduced binding by about 2-fold, and mutation of the other eight residues--by more than 10-fold. Analysis of these data and the mapping of these mutations on the recently determined crystal structure of a fragment containing the RBD complexed to ACE2 (Li, F, Li, W, Farzan, M, and Harrison, S. C., submitted) suggested the existence of two hot spots on the S RBD surface, R426 and N473, which are likely to contribute significant portion of the binding energy. The finding that most of the mutations (23 out of 34 including glycosylation sites) do not affect the RBD binding function indicates possible mechanisms for evasion of immune responses.

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Figures

Figure 1

Figure 1

Expression and binding of soluble S fragments containing the RBD. A) Soluble S proteins concentrated using Ni-NTA agarose beads from the supernatants of 293 cells transfected with various constructs were run, blotted onto a nitrocellulose membrane and detected with anti-c-myc epitope antibody. B) Cell binding assay data using supernatants described above, shown as a percentage of the reading of S272–537 that has been used in this experiment as a positive control.

Figure 2

Figure 2

Glycosylation of S fragment containing the RBD. A) Expression of the three mutants on S317–518 where the potential sites of glycosylation at N318, N330 and N357 were individually converted to alanine. All the mutants appear to have similar molecular weights when compared to the wild type protein S317–518. B) Cell binding data of the same mutants.

Figure 3

Figure 3

Effects of glycosylation on expression and binding of RBD-containing fragments. A) Expression of the four mutants on S319–518 where the two sites of glycosylation at N330 and N357 have been individually converted to either alanine or glutamine. The various mutants have similar molecular weights, a little less than the wild type indicating that the level of glycosylation at each residue might be similar. B) Cell binding data for the same mutants.

Figure 4

Figure 4

Glycosylation of at least one residue in RBD-containing fragments is required for expression. A) Expression pattern of two mutants on S319–518 in which both the glycosylation sites at N330 and N357 have been mutated either to alanine or to glutamine. No expression is seen when both the sites have been mutated indicating that glycosylation of at least one of the sites is important. In the last lane, purified S317–518 protein has been loaded as a control. B) Cell binding results of the same mutants.

Figure 5

Figure 5

Multiple sequence alignment of S fragment (RBD) with SARS CoV-related and other coronaviruses/spike glycoproteins. A) The table shows 13 amino acid residues in the region of S RBD (319–518) which have sequence variations as identified from the multiple sequence alignment of S RBD with 19 SARS CoV-related sequences (97–99% identities with S RBD) using BLAST. B) Multiple sequence alignment of S RBD and 7 other related proteins from different organisms which share 20–35% identities: bovine coronavirus (BCoV, 327–622), canine respiratory coronavirus (CCoV, 327–622), human coronavirus (OC43, 331–612), equine coronavirus (ECoV, 327–622), porcine hemagglutinating encephalomyelitis virus (PHEV, 327–608), rat sialodacryoadenitis coronavirus (RtCoV, 325–610) and murine hepatitis virus (MHV, 325–611). Dark and gray colors indicate the identity and similarity of residues aligned. Arrowheads on the S RBD sequence show the 13 sites, which are found to have sequence variations. C) The phylogram tree is shown with distances along the protein names and note that S RBD has the highest distance. Multiple sequence alignment and phylogram were constructed using ClustalW program.

Figure 6

Figure 6

Mapping of the S RBD mutants on the structure. The molecular surface diagrams of S RBD are shown as the top views in the solid and translucent models. The S RBD surface is in yellow, mutations that significantly affect the binding to ACE2 are in red and those do not affect the binding in cyan. (A) Shown are the solid surface diagrams using the structure of S RBD (left panel) and related by 180° rotations (right panel). The residues that decrease the receptor binding as observed in the experiment and exposed in the structure are labeled (R426, N473). (B) The same surface diagrams as in A but with transparency which are related by 180° rotations. The buried residues, which reduce the receptor binding as observed in the experiment, are seen as blurred red.

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