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Reconstitution of the receptor-binding motif of the SARS coronavirus.

ABSTRACT: The severe acute respiratory syndrome (SARS) coronavirus (CoV) identified in 2003 has infected ∼8000 people worldwide, killing nearly 10% of them. The infection of target cells by the SARS CoV is mediated through the interaction of the viral Spike (S) protein (1255 amino acids) and its cellular receptor, angiotensin-converting enzyme 2 (ACE2). The SARS CoV receptor-binding domain (amino acids N318-T509 of S protein) harbors an extended excursion along its periphery that contacts ACE2 and is designated the receptor-binding motif (RBM, amino acids S432-T486). In addition, the RBM is a major antigenic determinant, able to elicit production of neutralizing antibodies. Hence, the role of the RBM is a bi-functional bioactive surface that can be demonstrated by antibodies such as the neutralizing human anti-SARS monoclonal antibody (mAb) 80R which targets the RBM and competes with the ACE2 receptor for binding. Here, we employ phage-display peptide-libraries to reconstitute a functional RBM. This is achieved by generating a vast collection of candidate RBM peptides that present a diversity of conformations. Screening such 'Conformer Libraries' with corresponding ligands has produced short RBM constructs (ca. 40 amino acids) that can bind both the ACE2 receptor and the neutralizing mAb 80R.

SUBMITTER: Freund NT

PROVIDER: S-EPMC7107155 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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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Reconstitution of the receptor-binding motif of the SARS coronavirus.

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