Deamidation drives molecular aging of the SARS-CoV-2 spike protein receptor-binding motif

Abstract

The spike protein is the main protein component of the SARS-CoV-2 virion surface. The spike receptor-binding motif mediates recognition of the human angiotensin-converting enzyme 2 receptor, a critical step in infection, and is the preferential target for spike-neutralizing antibodies. Posttranslational modifications of the spike receptor-binding motif have been shown to modulate viral infectivity and host immune response, but these modifications are still being explored. Here we studied asparagine deamidation of the spike protein, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, which affect both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike protein. Asparagine residues 481 and 501 in the receptor-binding motif deamidate with a half-life of 16.5 and 123 days at 37 °C, respectively. Deamidation is significantly slowed at 4 °C, indicating a strong dependence of spike protein molecular aging on environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the human angiotensin-converting enzyme 2 receptor more than 3.5-fold, yet its high conservation pattern suggests some positive effect on viral fitness. We propose a model for deamidation of the full SARS-CoV-2 virion illustrating how deamidation of the spike receptor-binding motif could lead to the accumulation on the virion surface of a nonnegligible chemically diverse spike population in a timescale of days. Our findings provide a potential mechanism for molecular aging of the spike protein with significant consequences for understanding virus infectivity and vaccine development.

Keywords: RNA virus; SARS-CoV-2; molecular aging; protein deamidation; protein evolution; protein–protein interaction; receptor binding; receptor structure–function; spike.

Figure 1

Deamidation profile of S from Betacoronavirus. A, schematic representation of the deamidation reaction mechanism. B, deamidation profile of S proteins from a group of betacoronaviruses obtained with NGOME-LITE; only nonglycosylable asparagines are included. Deamidation half-times are shown on logarithmic scale. The Asn positions are referred to the SARS-CoV-2 sequence. C, deamidation hotspots on the SARS-CoV-2 S protein are shown in red. Protomers are colored in different gray tones. The PDB 6zgg (24) was used for this analysis, which contains a furin-cleaved SARS-CoV-2 S trimer, with two RBDs in the down conformation (chains A and C) and one in the up conformation (chain B). D, top view of (C), showing exposed deamidation hotspots at the RBD (Asn 481 and 501). E, zoom of the exposed Asn 544 residues.

Figure 2

Experimental deamidation kinetic and affinity to hACE2 of an aged RBD sample. A, left, time-decay of asparagine-containing peptides for the 481, 501, and 544 hotspots at 4 °C. The lines are fit to an exponential function with initial amplitude of 100% and endpoint of 0%. Right, time-decay of asparagine-containing peptides for the 481, 501, and 544 hotspots at 37 °C. The lines are fitted to an exponential decay. B, left, biolayer interferometry response curves of a fresh RBD sample to an immobilized hACE2. Right, biolayer interferometry response curve of an aged RBD sample (20 days at 37 °C). RBD, receptor-binding domain.

Figure 3

Conservation pattern of deamidation hotspots at the RBM of Sarbecoviruses. A, conservation of asparagines in SARS-Cov-2 shown as the percentage of Asn present in 38 sarbecoviruses versus deamidation half-times (Dataset S3). Hotspots 544, 856, and 907, red dots; hotspot 481, blue dot; and hotspot 501, green dot. B, alignment of different S proteins from selected sarbecoviruses. The RBM region is indicated, and the deamidation hotspots are highlighted in yellow. Residues of SARS-CoV and SARS-CoV-2 located closer than 5 Å from hACE2 residues are highlighted in cyan. C, location of the deamidation hotspots 481, 493, and 501 at the surface of SARS-CoV-2 RBD (pdb: 6M0J). Deamidation hotspots are shown in red, RBM (residues 438–506) is shown in ocher, and the core of the RBD is depicted in gray. hACE2 residues that directly interact with the RBM deamidation hotspots 493 (K31, H34, and E35) and 501 (Y41, K353, and D355) are shown as sticks. In SARS-CoV-2, the position 493 is a glutamine. D, superposition of RBD from SARS-CoV-2 (pdb 6M0J, pale blue) and an ensemble of modeled structures of RBD from bat-CoV Rs/YN2018A (gray). Asn 481 residue in SARS-CoV-2 RBD is shown in magenta sticks, and the Asn residues corresponding to the predicted 487 deamidation hotspot in Rs/YN2018A are drawn in red sticks. The inset details the receptor binding ridge (RBR) region. RBD, receptor-binding domain; RBM, receptor-binding motif.

Figure 4

Spike protein hotspot deamidation in the context of the SARS-CoV2 virion at 37 °C. A, simulated time course of the number of trimers with zero to six deamidation events at sites Asn 481 and Asn 501, using the deamidation half-times from Figure 2 (Fig. S3, for simulation details). We report the average and standard deviation of 1000 simulations using the Gillespie algorithm (Fig. S4 for full results and Table S8 for the results at several time points of interest). B, visualization of S protein deamidation in the SARS-CoV2 virion 48 h after synthesis. A cross section of the virus is shown (see (22) for details), with membrane and membrane-bound viral proteins M and E in green and the viral genome and associated nucleocapsid proteins N in blue. A total of 17 of the 33 spike trimers are shown, colored according to the simulation results (Table S8). Yellow, undeamidated spike monomers. Red, spike monomers deamidated at Asn 481. Orange, spike monomers deamidated at Asn 501.