Control state: no heat-treatment.
The workflow to include glycopeptides in HDX-MS analysis was then tested
after inducing a substantial change in spike structure by comparing
native and heat treated glycoprotein. Without heat treatment, spike
ectodomain (glyco)peptides showed a range of D2O
labeling (interpreted as structural dynamics) between
~0% and ~37% that visually correlated
with sub-domain location (Figure 6A). Localization of these patterns on
a model based upon Protein Data Bank (PDB) files 6VSB and 6VXX (55)
included key domains (furin cleavage site, fusion peptide, 630 loop,
etc.) often missing from RCSB structures
(www.rcsb.org), possibly because disordered
regions of spike can have low electron density in cryo-EM analysis (56).
The least deuterated area (<5%) was the trimeric core
interface (764-782 helix,
997-1020 helix, and 1043-1062 strand in β-sheet), as previously
described (4, 13).
The most deuterated regions (37%) were the “hinge” between head and
stalk regions (1132-1145), furin cleavage site (672-690), and 630 loop
(624-636), the latter also consistent with a previous report (13).
Labeling of spike’s “hinge” is a novel HDX-MS result, completely based
on the coverage of deuterated glycopeptides containing glycan-modified
N1134 (Figures 6 and S2). Robust labeling (24-30%) was observed in the
fusion peptide (823-851), N-terminus (19-31), and middle helix of the
stalk (1174-1197).
The receptor binding domain (RBD) had the highest deuteration (27%) on
a strand forming its hinge with the rest of S1 (319-346, including
deuterated glycopeptide coverage at N343) and the two loops of the
receptor binding motif (RBM, 442-452 and 471-487) for ACE2 (57). The
other strand forming the RBD’s hinge with S1 (516-533) was 20%
deuterated. A central helix (784-795), a turn-helix-turn (939-977) and a
helix U-turn (753-759) were also moderately deuterated (28-30%). The
N-terminal domain (NTD) showed an outer segment (171-176) with moderate
labeling (27%).
Several recent reports describe bimodality in isotopic envelopes for
certain regions of spike (1, 13, 15); the only apparently bimodal
peptide 878-904 in this study was interpreted to be chromatographic
co-elution of two precursor peptides with the same charge state and
overlapping precursor isotopic envelopes based on observing
“bimodality” even at 0 sec (data not shown).