Journal Article

Enhanced stability of the SARS CoV-2 spike glycoprotein following modification of an alanine cavity in the protein core

Ha, Jungmin, Zakir, Tasnim, Coulibaly, Fasséli, Center, Rob J., Aggarwal, Anupriya, Turville, Stuart G., Johnstone, Bronte A., Akerman, Anouschka, Langer, Christine, Drummer, Heidi E., Boo, Irene, Poumbourios, Pantelis, Milogiannakis, Vanessa

2023

Overview

The spike (S) glycoprotein of SARS CoV-2 is the target of neutralizing antibodies (NAbs) that are crucial for vaccine effectiveness. The S1 subunit binds ACE2 while the S2 subunit mediates virus-cell membrane fusion. S2 is a class I fusion glycoprotein subunit and contains a central coiled coil that acts as a scaffold for the conformational changes associated with fusion function. The coiled coil of S2 is unusual in that the 3–4 repeat of inward-facing positions are mostly occupied by polar residues that mediate few inter-helical contacts in the prefusion trimer. We examined how insertion of bulkier hydrophobic residues (Val, Leu, Ile, Phe) to fill a cavity next to Ala 1016 and Ala 1020 in the 3–4 repeat affects the stability and antigenicity of S trimers. Substitution of Ala 1016 with bulkier hydrophobic residues in the context of a prefusion-stabilized S trimer, S2P-FHA, was associated with increased thermal stability. S glycoprotein membrane fusion function was retained with Ala 1016 /Ala 1020 cavity-filling mutations associated with improved recombinant S2P-FHA thermostability, however 2 mutants, A1016L and A1016V/A1020I, lacked ability to mediate entry of S-HIV-1 pseudoparticles into 293-ACE2 cells. When assessed as immunogens, two thermostable S2P-FHA mutants derived from the ancestral isolate, A1016L (16L) and A1016V/A1020I (VI) elicited neutralizing antibody with 50%-inhibitory dilutions (ID 50 s) in the range 2,700–5,110 for ancestral and Delta-derived viruses, and 210–1,744 for Omicron BA.1. The antigens elicited antibody specificities directed to the receptor-binding domain (RBD), N-terminal domain (NTD), fusion peptide and stem region of S2. The VI mutation enabled the production of intrinsically stable Omicron BA.1 and Omicron BA.4/5 S2P-FHA-like ectodomain oligomers in the absence of an external trimerization motif (T4 foldon), thus representing an alternative approach for stabilizing oligomeric S glycoprotein vaccines.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Alanine / Amino acids / Angiotensin-Converting Enzyme 2 / Antibodies / Antibodies, Neutralizing / Antigenicity / Antigens / Biology and Life Sciences / Cell fusion / Cell membranes / Chromatography / Coils / Coronaviruses / COVID-19 / COVID-19 vaccines / Disease transmission / Domains / Glycoproteins / Health aspects / HIV / Human immunodeficiency virus / Humans / Hydrophobicity / Infections / Medicine and Health Sciences / Membrane fusion / Mutants / Mutation / Neutralizing / Oligomers / Pandemics / Peptides / Physiological aspects / Proteins / Research and Analysis Methods / Residues / Severe Acute Respiratory Syndrome / Severe acute respiratory syndrome coronavirus 2 / Spike glycoprotein / Spike Glycoprotein, Coronavirus / Structure / Thermal stability / Trimers / Vaccine efficacy / Vaccines / Viral antibodies / Viral diseases / Viruses