State difference (heat-treatment minus control).
Visualization of (heat – control) deuteration (Figure 6B), showed both increased and decreased labeling in different sub-domains of spike. Regions with no significant deuteration difference (within the ± 0.9 Da error of labeling estimation based upon average peptide length and repeatability, Table S2) were not interpreted to have “zero dynamic change” (42). Deuterated glycopeptide data provided coverage essential for revealing increased labeling with longer D2O exposure times around sequons N61 (11-15%) and N234 (11-15%) in the NTD, N343 in the RBD (10-15%), N603 (14-19%) and N801 (9%) in the S2 domain, as well as N1074 (10-16%) and N1134 (13-6%) in the stalk. Interestingly, only N1134 glycopeptides in the stalk region showed diminishing differences from the control state with increasing D2O exposure.
The region with lowest deuteration in the control state (trimer core interface) showed substantial increases in deuteration (764-782 helix, 21%, 1007-1024 helix, 20%, 1050-1062 β-strand, 20%). Regions with the highest deuteration in the control state showed only small increases [the “hinge” between head and stalk (1132-1145, 6%), the 630 loop (624-635, 5%)] or even reductions [the furin site (672-703, -8%) and fusion peptide (823-851, -2%)]. Data for changes in the “hinge” region were entirely from deuterated glycopeptides.
Increases in the deuteration of the RBD were interpreted as loosening of its sub-domain architecture, including outer loop 336-350 (10-24%) and the inner β-strand 393-399 (27%). In addition, the “hinge” of the RBD to S1 (515-533) and a loop of its RBM (442-453) had lower deuteration (-4%) after heat treatment. Furthermore, S2 domain helices interpreted to be dynamic and primed for fusion based on their substantial deuteration in the control state were lower (939-945 and 967-977, -5%) after heat treatment. Finally, the NTD showed spike’s highest reduction in deuteration (-10%) after heat-treatment at its outermost tip (243-262). All these results are consistent with increased accessibility to limited trypsin digestion (Figure S1), looser inter-monomer interaction, and disruption of tight packing between 3 NTDs and 3 RBDs from heat treatment. Reductions in deuteration observed for the most labeled sub-domains in the control state (furin site as an example) were interpreted as spike glycoprotein shriveling or collapsing after heat treatment.