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.