Available via license: CC BY 4.0
Content may be subject to copyright.
Evolution of the SARS-CoV-2 spike protein in the
human host
Antoni Wrobel ( antoni.wrobel@crick.ac.uk )
The Francis Crick Institute https://orcid.org/0000-0002-6680-5587
Donald Benton
Francis Crick Institute https://orcid.org/0000-0001-6748-9339
Chloë Roustan
The Francis Crick Institute
Annabel Borg
The Francis Crick Institute
Saira Hussain
The Francis Crick Institute
Stephen Martin
Structural Biology Science Technology Platform, the Francis Crick Institute, Mill Hill Laboratory, London,
UK.
Peter Rosenthal
The Francis Crick Institute https://orcid.org/0000-0002-0387-2862
John Skehel
Francis Crick Institute
Steve Gamblin
The Francis Crick Institute https://orcid.org/0000-0001-5331-639X
Biological Sciences - Article
Keywords: SARS-CoV-2, virology, microbiology
DOI: https://doi.org/10.21203/rs.3.rs-535704/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
1
Evolution of the SARS-CoV-2 spike protein in the
1
human host
2
3
Antoni G. Wrobel#*1, Donald J. Benton#*1, Chloë Roustan2, Annabel Borg2, Saira
4
Hussain3,4, Stephen R. Martin1, Peter B. Rosenthal5, John J. Skehel1, and Steven J.
5
Gamblin*1
6
7
1Structural Biology of Disease Processes Laboratory, 2Structural Biology Science
8
Technology Platform, 3Worldwide Influenza Centre, 4RNA Virus Replication
9
Laboratory, 5Structural Biology of Cells and Viruses Laboratory; Francis Crick Institute,
10
NW1 1AT London, United Kingdom
11
# equal contribution
12
*to whom correspondence should be addressed: antoni.wrobel@crick.ac.uk,
13
donald.benton@crick.ac.uk, steve.gamblin@crick.ac.uk
14
15
Variants of SARS-CoV-2 have emerged which contain multiple substitutions in
16
the surface spike glycoprotein that have been associated with increased
17
transmission and resistance to neutralising antibodies and antisera. We have
18
examined the structure and receptor binding properties of spike proteins from
19
the B.1.1.7 (UK) and B.1.351 (SA) variants to better understand the evolution of
20
the virus in humans. Both variants’ spikes have the same mutation, N501Y, in
21
their receptor-binding domains that confers tighter ACE2 binding and this
22
substitution relies on a common earlier substitution (D614G) to achieve the
23
tighter binding. Each variant spike has also acquired a key change in structure
24
that impacts virus pathogenesis. Unlike other SARS-CoV-2 spikes, the spike
25
from the UK variant is stable against detrimerisation on binding ACE2. This
26
feature primarily arises from the acquisition of a substitution at the S1-S2 furin
27
site that allows for near-complete cleavage. In the SA variant spike, the presence
28
of a new substitution, K417N, again on the background of the D614G
29
substitution, enables the spike trimer to adopt fully open conformations that are
30
required for receptor binding. Both types of structural change likely contribute
31
to the increased effectiveness of these viruses for infecting human cells.
32
33
2
The SARS-CoV-2 spike glycoprotein is the major surface antigen of the virus. Its
34
function is to bind the host receptor ACE2 and mediate the subsequent membrane
35
fusion required for cell entry 1–8. The virus has evolved in the human host during the
36
pandemic9–11 and we and others have demonstrated that the predominant D614G
37
substitution, located on a monomer-monomer interface of the spike trimer, increases
38
its ability to adopt the open conformation that is competent to bind receptor12–14.
39
Recently emerging variants of SARS-CoV-2 have acquired a number of other
40
substitutions in the spike including those on monomer-monomer interfaces, at the
41
receptor-binding site, and proximal to the furin-cleavage site (Fig. S1). Here we have
42
examined the structure and receptor binding properties of the B.1.1.7 variant first
43
described in Kent, United Kingdom (UK)10,15–18 and the B.1.351 variant first described
44
in South Africa (SA)19,20. The results provide a molecular explanation for the increased
45
human transmissibility of the variants.
46
First, examination of the structure of ACE2-bound spike protein of the UK variant
47
shows that all of the spikes are present as trimers (Fig. 1a, Supp. Table 1). This is
48
the first cleaved SARS-CoV-2 spike protein in complex with ACE2 we have observed
49
to be fully trimeric. All other spike structures, when mixed with ACE2, show a
50
substantial proportion of the material dissociated into monomers5. For example, in our
51
previous study of the furin-cleaved spike of the original Wuhan strain (Wuhan)
52
complexed with ACE2 (Fig. 1a), we observed that more than 70% of the particles
53
present were monomeric S/ACE2 complexes5. Notably, we also observed that this
54
spike material from the UK variant is almost fullly cleaved into S1 and S2 (Fig. S2),
55
which has also not been reported before8. This observation is consistent with the fact
56
that one of the mutations in the UK spike is the substitution P681H at the furin site
57
which generates a more basic cleavage site (HRRAR).
58
To test the possibility that it is the near complete cleavage of the S1-S2 subunits that
59
is responsible for the greater stability of the trimeric ACE2 bound S1/S2 complex, we
60
expressed the UK spike in the presence of a furin inhibitor, decanoyl-Arg-Val-Lys-Arg-
61
chloromethylketone, which resulted in a completely uncleaved protein (Fig. S2).
62
Incubation of this uncleaved spike with ACE2 resulted in more than 50% of particles
63
being monomeric S1-S2/ACE2 (Fig. 1a, S3). These data support the conclusion that
64
3
the near complete cleavage at the variant furin site enables spike to remain stable as
65
a trimer with ACE2 bound.
66
To better understand this phenomenon, we compared monomer/ACE2 complexes
67
from a number of SARS-CoV-2 variants (Figs. S4-5) and found that they all adopt a
68
similar overall conformation that, as noted before5, is markedly different from the
69
conformation adopted by the S/ACE2 complex in the trimeric species. As shown in
70
Fig. 1b, the monomeric and trimeric ACE2 complexes can be aligned very closely on
71
the ACE2/RBD components (coloured rosy brown in the figure) but, the NTD-
72
associated subdomain (light blue) and the NTD (blue) are rotated by 95° and their
73
centre of mass translated by 25 Å in the monomer, resulting in a conformation
74
incompatible with the trimer.
75
76
The fact that fully cleaved UK variant spike is able to remain stable as a trimer when
77
ACE2 bound indicates that the strain induced by receptor binding to RBD5 is relieved
78
if cleavage has occured between the S1 and S2 subunits. The cleavage site lies at the
79
C-terminus of the NTD-subdomain (shown on Fig. 1b), the individual centre of mass
80
of which is translocated 38 Å in the monomer/ACE2 complex compared to the fully
81
ACE2-bound trimer. Cleavage appears to be able to release sufficient strain to allow
82
the resulting ACE2 complex to remain stable as a trimer. The preponderance of
83
monomer S/ACE2 species in other SARS-CoV-2 variants, such as Wuhan (Fig. 1a)
84
and SA (Fig. S4), is probably accounted for by the lower levels of cleavage of these
85
spikes (Fig. S2), although our structures of unbound, uncleaved UK spike (Fig. S5-6)
86
suggest the trimeric state of the receptor-bound form of the UK variant spike might be
87
further stabilised by the substitutions D1118H and A570D on the inter-monomer
88
interfaces (Fig. S5). The increased stability of the receptor-bound UK spike may
89
enhance virus infectivity by increasing the avidity of binding to cells and by the priming
90
mechanism, which we have previously described5, in which ACE2 binding exposes
91
the S2 subunit for helical rearrangements associated with membrane fusion.
92
93
Second, we studied binding of ACE2 to spike variants by surface biolayer
94
interferometry. The data (Fig. 2a, b; Fig. S7) show a six-fold increase in binding
95
strength for UK spike, and a two-fold increase for SA spike (compared to Wuhan)
96
arising from the shared substitution N501Y in the RBD. The substitution of the
97
4
asparagine at position 501 in Wuhan for a tyrosine residue in both UK & SA variants
98
(Fig. 2c) leads to an increase in hydrophobic interactions between the aromatic ring
99
of Y501(RBD) and the aromatic ring of Tyr-41(ACE2) and the aliphatic moiety of Lys-
100
353(ACE2), in addition to a charged hydrogen bond between the phenolic hydroxyl of
101
Tyr-501(RBD) and Lys-353(ACE2) (Fig. 2d). Consistent with the smaller increase in affinity
102
for ACE2 of the SA spike versus UK spike, is the finding that, whereas UK has retained
103
the same salt bridge between Lys-417(RBD) and Asp-30(ACE2) as Wuhan, the RBD in SA
104
has acquired the additional substitution of an asparagine at RBD residue 417 which
105
cannot make this salt bridge (Fig. 2e).
106
107
To understand the evolution of receptor binding, we also expressed UK spike with an
108
aspartic acid, rather than glycine residue, at position 614. The substitution D614G
109
(relative to Wuhan) occurred earlier in the evolution of SARS-CoV-2, became the
110
predominant global form of the virus9 and continues to be present in the UK and SA
111
variant forms of the virus. Remarkably, the engineered G614D UK spike (Y501, D614)
112
shows the same binding affinity as Wuhan (N501, D614) (Fig. 2a,b; Fig. S7). Thus,
113
the additional binding energy, generated by the substitutions at the receptor-binding
114
interface, is only realised if residue 614 is also switched from aspartic acid to glycine.
115
We have previously shown that the D614G substitution leads to destabilisation of the
116
closed conformation thus promoting spike opening for subsequent receptor binding
117
and membrane fusion12. This occurs when residue 614 is an aspartic acid (on the S1
118
NTD-subdomain) and it creates a salt bridge with Lys-854 (on a neighbouring S2
119
subunit) stabilising the closed form of the trimer5,12,21. Mutation to glycine at residue
120
614 precludes this interaction and thus disfavours the closed-form of spike. We have
121
previously envisaged a low energy barrier to the closed to open transition and the
122
present data show that the G614 (open) form of spike is necessary for the Y501
123
substitution to produce tighter binding.
124
125
Third, the most striking feature of the structure of SA spike revealed by cryoEM is that
126
all the trimers adopt an open conformation with either one (64%) or two (36%) of the
127
RBDs in the erect, receptor-binding-competent form (Fig. 3a, Fig S6). This
128
observation is in marked contrast to the earliest SARS-CoV-2 viruses where 83% of
129
Wuhan spike particles were in the closed form (Fig. 3a)8 and is noteworthy with
130
5
regards to the subsequent D614G form of the protein where we observed 87% of
131
spikes in an open conformation, out of which only 18% were in the two-RBD-erect
132
conformation (Fig. S6)12. Inspection of the sequence of the SA spike and comparison
133
of its structure with that of the closed form of Wuhan spike and the open and closed
134
forms of G614 spikes, suggests that the complete opening of SA spike is driven by the
135
substitution K417N on the background of G614. In Wuhan, Lysine at residue 417 on
136
the RBD not only makes an aliphatic packing interaction with Tyr-369 of the
137
neighbouring subunit, it also forms a salt bridge/hydrogen bond network with Glu-406
138
and Arg-403 of the RBD and with Ser-373 of the neighbouring RBD that stabilises the
139
closed conformation. In contrast, the substitution of an asparagine at position 417 in
140
SA removes an intramolecular salt-bridge and would generate a steric clash with Tyr-
141
369 of the neighbouring RBD leading to destabilisation of the closed form and thus
142
promotion of the open form (Fig. 3b). In the same way that G614 is a prerequisite for
143
realising tighter receptor binding by the substitution N501Y described above, it likely
144
also enables spike protein from the SA virus to achieve a fully open conformation as
145
a result of the K417N substitution.
146
147
We showed before that the extent of cleavage at the furin site influences the proportion
148
of spike molecules being open and thus able to bind receptor8. We and others also
149
showed that the D614G substitution in the spike, which was acquired early in the
150
pandemic, similarly acted to increase the proportion of open forms of spike12–14. We
151
now show that the emergence of UK spike, which is completely cleaved, and SA spike,
152
which does not adopt the closed conformation, represent two related additional steps
153
in viral adaptation to the human host. Modifications in the spike glycoprotein during
154
evolution of the SARS-CoV-2 virus in humans therefore have made the virus more
155
infectious by increasing the stability of trimeric spike, by tighter receptor binding, and
156
by promotion of the open forms of spike. Although as yet uncharacterised at the
157
molecular level, the variant of concern B.1.61722,23, which recently emerged in India,
158
contains the substitution P681R which we suspect will have a similar impact on the
159
virus’ spike structure, and thus its pathogenesis, as the P681H substitution in the UK
160
variant.
161
162
6
163
164
Figure 1: Structures of UK variant spike interacting with ACE2 receptor. (A) Surface
165
representations of predominant molecular species present when ACE2 is mixed with the UK
166
variant spike expressed in absence (left panel) or presence (middle panel) of furin inhibitor I,
167
compared to the predominant species when ACE2 is mixed with Wuhan spike from our
168
previous study (right panel) (PDB ID 7A91 5). ACE2 is coloured in green, with monomers of
169
the spike coloured in blue, goldenrod, and rosy brown in the middle panel and rosy brown for
170
S monomers in the right and left panels. (B) Comparison of the domain movements of S
171
complexed to ACE2 in its trimeric (middle) compared to monomeric (left) form with (right) the
172
detail showing the location of the unstructured loop between the NTDG and S2, where the
173
cleavage site is located. Domains are coloured: RBD in rosy brown, RBD-associated
174
subdomain (ganymede, RBDG) in pink, NTD ganymede (NTDG) in light blue, NTD in navy,
175
and S2 in red.
176
177
7
178
Figure 2: Variant spike binding to receptor ACE2. (A) Kd of variant spikes binding
179
to ACE2 measured using biolayer interferometry and calculated from koff/kon analysis
180
(see Fig. S7 for details). (B) Plots of fractional saturation binding measurements with
181
data for the UK variant showed in red, UK variant with G614D substitution in pink, and
182
Wuhan (D614) spike in blue. Wuhan (*the data for which shown here are adapted from
183
our previous work24) and UK D614 spike show almost identical affinity towards ACE2.
184
Similar results were obtained for G614 vs D614 mink (Y453F) spike (Fig. S4, S7). (C)
185
cryoEM density of the complex of SA variant S1 with ACE2, with ACE2 coloured in
186
green, RBD in rosy brown, RBD subdomain in plum, with the remaining S1
187
disseminated density in cream. (D, E) Detail of changes in the binding interfaces
188
present in variants (left column) compared to the Wuhan strain (right column). (D) The
189
N501Y substitution present in both the SA and UK variants allows formation of a new
190
hydrogen bond or a salt bridge. (E) The K417N substitution present in the SA variant
191
eliminates a salt bridge between the RBD and ACE2.
192
193
8
194
195
Figure 3: South African variant spike trimer structures. (A) (Left and centre)
196
Surface representations of cryoEM structures of the SA variant spike, with monomers
197
coloured in blue, goldenrod and rosy brown, compared to (right) the closed form of the
198
Wuhan strain determined in our previous study (PDB ID 6ZGE 8) shown in paler hues
199
of the same colours. The SA variant adopts a fully open conformation. (B) The K417N
200
substitution present in the SA variant likely destabilises the closed form of the protein
201
by introducing a steric clash and interfering with the network of electrostatic
202
interactions present at the trimer interface in the closed form of the Wuhan protein.
203
Notably, R417 in the fully-closed Pangolin-CoV spike seems to stabilise the same
204
network of RBD-RBD interactions24.
205
206
207
9
Methods
208
209
Construct design
210
The SARS-CoV-2 spike constructs used in this study were derived by Genscript from
211
the spike ectodomain (residues 1-1208) constructs of the Wuhan variant cloned into
212
pcDNA.3.1(+) described before5,8. The variant spikes were stabilised in the pre-fusion
213
conformation (K986P and V987P)25 with the furin-cleavage site ([PH]RRAR) either
214
intact (‘2P’ constructs) or mutated to the uncleavable sequence PSRAS (‘FUR2P’
215
constructs). The following variant spikes were made (SA19, UK18 and mink26) (all
216
mutations listed with reference to the NCBI sequence YP_009724390.1): 2P UK (Δ69-
217
70, Δ144, N501Y, A570D, D614G, P681H, T716A, S982A, D1118H, and K986P,
218
V987P), FUR2P UK (Δ69-70, Δ144, N501Y, A570D, D614G, T716A, S982A, D1118H,
219
and R682S, R685S, K986P V987P), FUR2P D614 UK (Δ69-70, Δ144, N501Y, A570D,
220
T716A, S982A, D1118H, and R682S, R685S, K986P, V987P), 2P SA spike (L18F,
221
D80A, D215G, R246I, K417N, E484K, N501Y, D614G, A701V, and K986P, V987P),
222
FUR2P SA spike (L18F, D80A, D215G, Δ242-244, R246I, K417N, E484K, N501Y,
223
D614G, A701V, and R682S, R685S, K986P, V987P), FUR2P mink (Δ69-70, Y453F,
224
D614G, I692V, and R682S, R685S, K986P, V987P), FUR2P D614 mink (Δ69-70,
225
Y453F, I692V, and R682S, R685S, K986P, V987P). The Wuhan spikes and ACE2
226
ectodomain construct (residues 19-615) used in this study were exactly as described
227
before5,8.
228
229
Protein expression and purification
230
All the variant spikes were expressed in suspension-cultured Expi293F cells (Gibco)
231
cells and purified as described before for the D614G spike12. In brief, Expi293F cells
232
were cultured at 37°C, shaking at 125 rpm, in humidified 8% CO2 atmosphere in
233
FreeStyle 293 Expression Medium and transfected with 1 mg of spike DNA per litre of
234
culture at cell density of 3*106/mL. For expression in presence of a furin inhibitor,
235
decanoyl-Arg-Val-Lys-Arg-chloromethylketone (also known as “furin inhibitor I”) was
236
prepared as a 23.5 mg/mL stock solution in DMSO and added to the cells at final
237
concentration of 100 µM half an hour prior to transfection. Next day after transfection,
238
the cells were enhanced according to the manufacturer’s instructions and transferred
239
to 32°C27.
240
10
The supernatant was harvested on the fifth day post-transfection and the spike purified
241
using cobalt NTA beads (TAKARA). The protein was then eluted in PBS with 200 mM
242
imidazole, concentrated, and either flash-frozen or gel filtered at room temperature
243
into a buffer containing 150 mM NaCl, TRIS pH 8 on a Superdex 200 Increase 10/300
244
GL column (GE Life Sciences). Wuhan spikes and ACE2 were made exactly as
245
described before5,8.
246
247
Biolayer interferometry
248
Measurements of spike variants affinity towards human ACE2 ectodomain were
249
performed at 25°C on an Octet Red 96 instrument (ForteBio) with shaking at 1000 rpm
250
in 150 mM NaCl, 20 mM TRIS pH 8. First, variant spikes at 40-80 µg/mL were
251
immobilised for 40-60 minutes on NiNTA sensors pre-equilibrated in buffer. Then, the
252
ACE2 binding was measured using 2-5 minutes association and 10-20 minutes
253
dissociation phases. At least three independent measurements were made for each
254
spike.
255
256
The data were analysed using kinetic and equilibrium methods. For equilibrium
257
analysis, the data were first normalised by dividing by the maximum observable
258
response in order to give fractional saturation as a function of ACE2 concentration.
259
The KD was then determined from analysis of the variation of fractional saturation with
260
ACE2 concentration. For kinetic analysis, plots of the observed rate (kobs) were derived
261
from association phases using a single exponential function and kon and koff were
262
obtained from plots of kobs vs ACE2 concentration as the slope and intercept
263
respectively.
264
265
Cryo-EM sample preparation
266
Samples were frozen on R2/2 400 mesh Quantifoil grids glow-discharged for 30 s at
267
25 mA. A sample at 0.5-0.8 mg/mL final concentration of the spike was supplemented
268
with 0.1% (final concentration) octyl glucoside, 4 µL of it applied on a grid, blotted for
269
5 s to 6 s with filter paper pre-equilibrated at 4˚C in 100% humidity, and plunge frozen
270
in liquid ethane using Vitrobot Mark III. In order to obtain ACE2-spike complexes the
271
proteins were mixed at 2 to 1 molar ratio of ACE2 to spike trimer and incubated at
272
room temperature for 20-40 minutes prior to grid freezing.
273
11
274
Cryo-EM data collection
275
Data were collected using EPU software (Thermo Scientific) on Titan Krios
276
microscopes operating at 300 kV either with a Falcon 3 camera (Thermo Scientific)
277
operating in electron-counting mode (60 s exposures, total dose of 35 e/Å2,
278
fractionated into 30 frames, 1.09 Å2 calibrated pixel size) or K2 camera (Gatan) with
279
GIF Quantum LS energy filter (Gatan) with a slit width of 20 eV operating in the zero-
280
loss mode (9.4 s exposures, 49 e/Å2 total dose, fractionated into 32 frames, 1.08 Å2
281
calibrated pixel size). The following eight datasets were collected (Supp. Table 1):
282
FUR2P SA (Falcon 3), ACE2 + FUR2P SA (K2), FUR2P UK (Falcon 3), ACE2 +
283
FUR2P D614 UK (K2), ACE2 + 2P UK furin-uninhibited (Falcon 3), ACE2 + 2P UK
284
furin-inhibited (Falcon 3), FUR2P mink (K2), ACE2 + FUR2P D614 mink (K2). All
285
micrographs were collected with defocus range between 1.5 and 3 µm.
286
287
Cryo-EM data processing
288
Collected movies were motion corrected using MotionCor228 implemented in
289
RELION29 and contrast transfer functions estimated using CTFind430. Particles were
290
picked using crYOLO31 using manually trained models. Particles were extracted 2x
291
downsampled in RELION before two rounds 2D classification in cryoSPARC32.
292
Classes which showed clear secondary structure were retained and an initial model
293
generated also using cryoSPARC. Particles from the selected classes were 3D
294
classified in RELION. The selection process for the different data collections are
295
shown in Figs. S3, S4, S6. Final particle stacks were re-extracted unbinned and
296
subjected to Bayesian polishing in RELION, and refined in cryoSPARC using either
297
Homogeneous refinement or Non-Uniform Refinement routines, both coupled to per-
298
particle defocus refinement. Final maps had their local resolution estimated using
299
blocres33 implemented in cryoSPARC (Fig. S8), followed by local resolution filtering in
300
cryoSPARC and b-factor sharpening34.
301
302
Model building, refinement, and validation
303
High resolution models of the monomeric S-ACE2 complexes were based on the
304
previously determined model for the non-uniform map refinement of the monomeric
305
Wuhan spike in complex with ACE2 (PDB 7A91)5. The low-resolution model of the
306
12
monomeric UK S-ACE2 complex was built from the non-uniform-refined, high
307
resolution model of the same protein from this study combined with the monomeric
308
Wuhan S-ACE2 (PDB 7A92)5. Models for the unbound trimeric spikes were based on
309
our model of D614G spike (PDB IDs 7BNM, 7BNN, 7BNO)12. The model of the three-
310
ACE2-bound trimer of the 2P UK spike was based on the three-ACE2 bound Wuhan
311
spike we determined before (PDB ID 7A98)5, in which the RBD (spike residues 333-
312
527) and ACE2 were replaced by those from the monomeric, non-uniformed-refined
313
UK S-ACE2 determined in this study. All structures were manually adjusted in
314
COOT35, refined with PHENIX Real Space Refine and validated in PHENIX36.
315
Measurements were performed with CCP4mg37 and Chimera38.
316
317
318
13
Author Contributions
319
A.G.W., C.R., D.J.B, S.H., S.R.M., performed research, collected and analysed data; A.G.W,
320
D.J.B, P.B.R, J.J.S, S.J.G conceived and designed research and wrote the paper.
321
Conflict Statement
322
We have no conflicts of interest to declare.
323
Data Availability
324
Maps and models have been deposited in the Electron Microscopy Data Bank,
325
http://www.ebi.ac.uk/pdbe/emdb/ (Accession numbers XXX). Models have been deposited in
326
the Protein Data Bank, https://www.ebi.ac.uk/pdbe/ (PDB ID codes XXX). [Accession numbers
327
will be available before publication].
328
Acknowledgements
329
We would like to acknowledge Andrea Nans of the Crick Structural Biology Science
330
Technology Platform for assistance with data collection, Phil Walker and Andrew Purkiss of
331
the Crick Structural Biology Science Technology Platform and the Crick Scientific Computing
332
Science Technology Platform for computational support. We thank Peter Cherepanov, George
333
Kassiotis, and Svend Kjaer for discussions. This work was funded by the Francis Crick Institute
334
which receives its core funding from Cancer Research UK (FC001078 and FC001143), the
335
UK Medical Research Council (FC001078 and FC001143), and the Wellcome Trust
336
(FC001078 and FC001143). The work done at the Crick Worldwide Influenza Centre, a WHO
337
Collaborating Centre for Reference and Research on Influenza, was supported by the Francis
338
Crick Institute receiving core funding from Cancer Research UK (FC001030), the Medical
339
Research Council (FC001030) and the Wellcome Trust (FC001030).
340
341
14
References
342
343
1. Walls, A. C. et al. Cryo-electron microscopy structure of a coronavirus spike
344
glycoprotein trimer. Nature (2016) doi:10.1038/nature16988.
345
2. Walls, A. C. et al. Tectonic conformational changes of a coronavirus spike
346
glycoprotein promote membrane fusion. PNAS (2017) doi:10.1073/pnas.1708727114.
347
3. Walls, A. C. et al. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike
348
Glycoprotein. (2020) doi:10.1016/j.cell.2020.02.058.
349
4. Wrapp, D. et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion
350
conformation. Science (80-. ). (2020) doi:10.1126/science.aax0902.
351
5. Benton, D. J. et al. Receptor binding and priming of the spike protein of SARS-CoV-2
352
for membrane fusion. Nature (2020) doi:10.1038/s41586-020-2772-0.
353
6. Cai, Y. et al. Distinct conformational states of SARS-CoV-2 spike protein. Science
354
(80-. ). eabd4251 (2020) doi:10.1126/science.abd4251.
355
7. Ke, Z. et al. Structures and distributions of SARS-CoV-2 spike proteins on intact
356
virions. Nature (2020) doi:10.1038/s41586-020-2665-2.
357
8. Wrobel, A. G. et al. SARS-CoV-2 and bat RaTG13 spike glycoprotein structures
358
inform on virus evolution and furin-cleavage effects. Nat. Struct. Mol. Biol. 1–5 (2020)
359
doi:10.1038/s41594-020-0468-7.
360
9. Korber, B. et al. Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G
361
Increases Infectivity of the COVID-19 Virus. Cell (2020)
362
doi:10.1016/j.cell.2020.06.043.
363
10. Challen, R. et al. Risk of mortality in patients infected with SARS-CoV-2 variant of
364
concern 202012/1: Matched cohort study. BMJ 372, (2021).
365
11. Davies, N. G. et al. Increased mortality in community-tested cases of SARS-CoV-2
366
lineage B.1.1.7. Nature 593, 270–274 (2021).
367
12. Benton, D. J. et al. The effect of the D614G substitution on the structure of the spike
368
glycoprotein of SARS-CoV-2. PNAS 118, (2021).
369
13. Zhang, J. et al. Structural impact on SARS-CoV-2 spike protein by D614G
370
substitution. Science (80-. ). 372, eabf2303 (2021).
371
14. Gobeil, S. M. C. et al. D614G Mutation Alters SARS-CoV-2 Spike Conformation and
372
Enhances Protease Cleavage at the S1/S2 Junction. Cell Rep. 34, 108630 (2021).
373
15. Peter Horby, Catherine Huntley, Nick Davies, John Edmunds, N. F. & Graham
374
Medley, C. S. NERVTAG - Presented to SAGE on 21/1/21.
375
https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachm
376
ent_data/file/961037/NERVTAG_note_on_B.1.1.7_severity_for_SAGE_77__1_.pdf.
377
16. Public Health England. Investigation of novel SARS-CoV-2 variant: Variant of
378
Concern 202012/01.
379
https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachm
380
ent_data/file/959360/Variant_of_Concern_VOC_202012_01_Technical_Briefing_3.pdf
381
.
382
17. Davies, N. G. et al. Estimated transmissibility and impact of SARS-CoV-2 lineage
383
B.1.1.7 in England. Science (80-. ). 372, eabg3055 (2021).
384
18. Rambaut, A. et al. Preliminary genomic characterisation of an emergent SARS-CoV-2
385
lineage in the UK defined by a novel set of spike mutations.
386
https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sars-cov-
387
2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563.
388
19. Tegally, H. et al. Emergence of a SARS-CoV-2 variant of concern with mutations in
389
spike glycoprotein. Nature 592, 438 (2021).
390
20. Tegally, H. et al. Sixteen novel lineages of SARS-CoV-2 in South Africa. Nat. Med.
391
27, 440–446 (2021).
392
21. Xiong, X. et al. A thermostable, closed SARS-CoV-2 spike protein trimer. Nat. Struct.
393
Mol. Biol. (2020) doi:10.1038/s41594-020-0478-5.
394
15
22. Threat Assessment Brief: Emergence of SARS-CoV-2 B.1.617 variants in India and
395
situation in the EU/EEA. https://www.ecdc.europa.eu/en/publications-data/threat-
396
assessment-emergence-sars-cov-2-b1617-variants.
397
23. Nervtag. SARS-CoV-2 variants of concern and variants under investigation in
398
England.
399
https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachm
400
ent_data/file/984274/Variants_of_Concern_VOC_Technical_Briefing_10_England.pdf
401
(2021).
402
24. Wrobel, A. G. et al. Structure and binding properties of Pangolin-CoV spike
403
glycoprotein inform the evolution of SARS-CoV-2. Nat. Comm. 12, 1–6 (2021).
404
25. Pallesen, J. et al. Immunogenicity and structures of a rationally designed prefusion
405
MERS-CoV spike antigen. PNAS 114, E7348–E7357 (2017).
406
26. Ecdc. Detection of new SARS-CoV-2 variants related to mink.
407
https://www.ecdc.europa.eu/sites/default/files/documents/RRA-SARS-CoV-2-in-mink-
408
12-nov-2020.pdf (2020).
409
27. Esposito, D. et al. Optimizing high-yield production of SARS-CoV-2 soluble spike
410
trimers for serology assays. Protein Expr. Purif. 174, 105686 (2020).
411
28. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for
412
improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
413
29. Scheres, S. H. W. RELION: Implementation of a Bayesian approach to cryo-EM
414
structure determination. J. Struct. Biol. 180, 519–530 (2012).
415
30. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from
416
electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
417
31. Wagner, T. et al. SPHIRE-crYOLO is a fast and accurate fully automated particle
418
picker for cryo-EM. Commun. Biol. 2, (2019).
419
32. Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms
420
for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296
421
(2017).
422
33. Cardone, G., Heymann, J. B. & Steven, A. C. One number does not fit all: Mapping
423
local variations in resolution in cryo-EM reconstructions. J. Struct. Biol. (2013)
424
doi:10.1016/j.jsb.2013.08.002.
425
34. Rosenthal, P. B. & Henderson, R. Optimal Determination of Particle Orientation,
426
Absolute Hand, and Contrast Loss in Single-particle Electron Cryomicroscopy. J. Mol.
427
Biol. 333, 721–745 (2003).
428
35. Emsley, P., Lohkamp, B., Scott, W. G., Cowtan, K. & IUCr. Features and development
429
of Coot. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 486–501 (2010).
430
36. Adams, P. D. et al. PHENIX : a comprehensive Python-based system for
431
macromolecular structure solution. Acta Crystallogr. Sect. D Biol. Crystallogr. 66,
432
213–221 (2010).
433
37. McNicholas, S., Potterton, E., Wilson, K. S. & Noble, M. E. M. Presenting your
434
structures: The CCP4mg molecular-graphics software. Acta Crystallogr. Sect. D Biol.
435
Crystallogr. 67, 386–394 (2011).
436
38. Pettersen, E. F. et al. UCSF Chimera - A visualization system for exploratory research
437
and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
438
439
16
Supplementary Table 1:
Cryo-EM data collection, refinement and validation statistics
UK
Monomer
+ACE2
(EMDB-
xxxx)
(PDB xxxx)
UK
Monomer
+ACE2
(with
accessories)
(EMDB-
xxxx)
(PDB xxxx)
SA
Monomer
+ACE2
(EMDB-
xxxx)
(PDB xxxx)
Mink
Monomer
+ACE2
(EMDB-
xxxx)
(PDB xxxx)
UK Trimer
Closed
(EMDB-
xxxx)
(PDB xxxx)
UK Trimer 1
Erect RBD
(EMDB-
xxxx)
(PDB xxxx)
UK Trimer 2
Erect RBD
(EMDB-
xxxx)
(PDB xxxx)
SA Trimer 1
Erect RBD
(EMDB-
xxxx)
(PDB xxxx)
SA Trimer 2
Erect RBD
(EMDB-
xxxx)
(PDB xxxx)
Mink
Trimer
Closed
(EMDB-
xxxx)
(PDB xxxx)
Mink
Trimer 1
Erect RBD
(EMDB-
xxxx)
(PDB xxxx)
Mink
Trimer 2
Erect RBD
(EMDB-
xxxx)
(PDB xxxx)
UK Trimer + 3
ACE2
(EMDB-xxxx)
(PDB xxxx)
Data collection and
processing
Voltage (kV)
300
300
300
300
300
300
300
300
300
300
300
300
300
Electron exposure (e–
/Å2)
49
49
49
49
35
35
35
35
35
49
49
49
35
Defocus range (μm)
-1.5 to -3.0
-1.5 to -3.0
-1.5 to -3.0
-1.5 to -3.0
-1.5 to -3.0
-1.5 to -3.0
-1.5 to -3.0
-1.5 to -3.0
-1.5 to -3.0
-1.5 to -3.0
-1.5 to -3.0
-1.5 to -3.0
-1.5 to -3.0
Pixel size (Å)
1.08
1.08
1.08
1.08
1.09
1.09
1.09
1.09
1.09
1.08
1.08
1.08
1.09
Symmetry imposed
C1
C1
C1
C1
C3
C1
C1
C1
C1
C3
C1
C1
C3
Final particle images
(no.)
187k
187k
121k
393k
50k
316k
46k
196k
111k
84k
602k
106k
45k
Map resolution (Å)
FSC threshold = 0.143
3.5
4.0
3.5
3.3
3.6
3.4
4.1
3.5
3.7
3.0
2.8
3.3
3.9
Refinement
Initial model used (PDB
code)
7A91
7A92
7A91
7A91
7BNM
7BNN
7BNO
7BNN
7BNO
7BNM
7BNN
7BNO
7A98
Model resolution (Å)
FSC threshold = 0.5
3.5
4.0
3.7
3.3
3.6
3.4
4.3
3.6
3.9
3.2
2.9
3.5
4.0
Map sharpening B factor
(Å2)
-130.2
-108.6
-132.7
-134.2
-140.0
-151.5
-123.0
-141.7
-132.8
-98.7
-103.0
-88.4
-119.0
Model composition
Non-hydrogen atoms
Protein residues
Ligands
6891
839
9
9823
1210
8
6870
837
8
6858
839
7
24303
3003
57
23655
2939
50
24109
3000
43
24067
3005
40
23641
2981
22
25773
3189
60
25562
3193
42
25400
3178
38
38670
4758
60
R.m.s. deviations
Bond lengths (Å)
Bond angles (°)
0.005
0.621
0.002
0.457
0.003
0.529
0.003
0.529
0.003
0.547
0.005
0.607
0.004
0.526
0.004
0.571
0.003
0.504
0.005
0.673
0.004
0.651
0.003
0.556
0.003
0.511
Validation
MolProbity score
Clashscore
Poor rotamers (%)
1.27
3.78
0.00
1.53
6.34
0.00
1.42
4.17
0.54
1.33
4.03
0.00
1.67
6.22
0.00
1.78
5.29
0.04
1.97
10.95
0.00
1.60
4.65
0.19
1.46
5.15
0.50
1.32
2.68
0.32
1.34
3.31
0.54
1.21
3.99
0.18
1.76
8.24
0.00
Ramachandran plot
Favored (%)
Allowed (%)
Disallowed (%)
97.47
2.53
0.00
96.96
3.04
0.00
96.62
3.38
0.00
97.23
2.77
0.00
95.32
4.68
0.00
91.81
8.19
0.00
93.85
6.15
0.00
94.80
5.20
0.00
96.88
3.12
0.00
96.07
3.93
0.00
96.64
3.36
0.00
97.90
2.10
0.00
95.50
4.50
0.00
17
Figure S1: Location of changes in spike protein of different SARS-CoV-2 variants
investigated in this study. Changes are highlighted on our previous structure of SARS-CoV-
2 spike in closed conformation (PDB ID 6ZGE 8) (left). Substitutions of particular interest–
located either on the intra-trimer interfaces or the RBD–are highlighted in red. Changes in the
NTD–mainly restricted to surface interfaces–are shown in either dark (substitutions) or light
(deletions) green. Other changes are highlighted in blue. A full list of changes is shown for
each variant on the right.
18
Figure S2: Cleavage state of variant spikes. (A, B) SDS PAGE analysis of purified spikes
used in this study. (A) From left, lanes contain: molecular weight markers (Precision Blue
Protein Standards, All Blue, Bio-rad), uncleavable (‘FUR2P’: R682S+R685S) Wuhan spike,
and 2P-only spikes: Wuhan, SA, UK, P681H-only (otherwise identical to the Wuhan 2P). UK
and P681H-only spikes are almost fully cleaved into S1 and S2 while SA and Wuhan spikes
are cleaved only partially; this indicates that the P681H substitution in the cleavage site is
sufficient to cause the extent of cleavage observed in the UK variant. (B) Cleavage state of
P681H-only (left two lanes) and UK (right two lanes) spikes expressed in presence of furin
inhibitor I (lanes “I”) compared to non-inibited (n-I). The inhibitor almost fully prevents spike
cleavage into S1 and S2. (C) Sequence alignment of the S1/S2 cleavage site from several
SARS-CoV-2 strains and related sarbecoviruses: SARS-CoV and the bat-CoV most closely
related to SARS-CoV-2, RaTG13. SARS-CoV-2 acquired furin-cleavage site RxxR during its
evolution and this site has become more polybasic (blue) in the recent UK and Indian variants.
19
Figure S3: cryoEM image processing scheme for UK variant in furin uninhibited (cleaved) and
furin inhibited (uncleaved) forms binding to ACE2.
20
Figure S4: cryoEM image processing scheme for UK (G614D), SA and Mink (G614D)
variants in complex with ACE2.
21
22
Figure S5: Structural insights into mink and UK variant spikes. (A) Surface
representations of mink spike structures determined in this study: in a dilated, closed
conformation different to that of the Wuhan spike but resembling D614G spike12; and one- and
two-RBD erect conformations, the latter of which is assumed by D614G but not by Wuhan
spike12. (B) Cryo-EM density of monomeric complex of D614 mink spike and ACE2. (C) Detail
of the mink spike / ACE2 interface (left) compared to Wuhan (right). The His-34, which adopts
two alternative conformations in Wuhan spike, is present only as one rotamer in the structure
of the mink spike/ACE2 complex (Fig. S4).
(D) Surface representations of furin-uncleavable UK spike in three conformations which
resemble those of the mink and D614G-only spike we described previously12: a dilated-closed
conformation different to that of the Wuhan variant and two open conformations, with one or
two RBDs erect.
(E, F) Detail of substitutions in UK spike (left panels) compared to Wuhan (right panels) that
may further contribute to enhanced trimeric state of the open and receptor-bound forms of UK
spike. (E) A570 in Wuhan strain lies close to the interface between RBD-associated
subdomain (RBDG, blue) and the fragment of S2 core of the neighbouring chain (residues
815-855, red), which undergoes significant rearrangement upon spike opening (the unfolded
region 824-853 is structured in the closed Wuhan spike). In (D570) UK spike the whole RBDG
domain shifts slightly closer towards S2 and the same region of the neighbouring-chain S2,
especially F855, adopts different conformation upon spike opening. This alternative
conformation might be attributed to a formation of a salt bridge between K854, which
incidentally makes a salt bridge with D614 in the original Wuhan strain but not in the later
G614 variants, and D570 in the UK variant spike. (F) The S2 residue 1118 lies at the
membrane proximal end of the UK spike trimer, close to the trimer axis. It is an aspartic acid
in Wuhan (right) but a histidine in UK. It appears that a cluster of trimer-related aspartic acid
side chains at this position forms a less stable arrangement than is be adopted by a
corresponding cluster of neutral histidine residues in the UK spike, which also coordinate a
smaller molecule or an ion-here modelled as water. These two substitutions could explain
greater stability of the open form of UK spike. Indeed, a comparison of the overall surface
areas of the monomer-monomer interfaces in the 1-RBD-erect conformations of the UK and
G614-only trimers shows that the former is 700 Å2 larger providing more evidence to why the
UK spike is less likely to undergo disassembly upon receptor binding than other variants.
23
Figure S6: cryoEM image processing scheme for spike trimers of UK, SA and Mink variants.
24
Figure S7: Biolayer interferometry binding measurements of ACE2 binding to
immobilised variant spikes. (A) Thermodynamic parameters for ACE2 binding to different
spikes. Association rate constants (kon) were determined from the slopes of plots of the
observed rate constant against ACE2 concentration shown in panel C. Dissociation rate
constants (koff) were determined from the intercepts of these plots and through independent
analysis of the dissociation phase. The Kd values were calculated from the kinetic data as
koff/kon (Kd(Kin)) and from analysis of the dependence of fractional saturation on ACE2
concentration shown in main Fig. 2b and panel B below (Kd(Amp)). (B) Variation of fractional
saturation with ACE2 concentration for different spikes. The solid lines are the computed best
fits. * The data for Wuhan (D614) shown here are adapted from our previous work (Pangolin
ref). (C) Dependence of the observed rate constant (kobs) on ACE2 concentration for different
spikes.
25
Figure S8: Fourier Shell Correlation (FSC) curves for each of the deposited structures.
Figures
Figure 1
TG / DTG curves of coal sample at different heating rates
Figure 2
Ionic strength of coal pyrolysis gas at 10 °C/ min
Figure 3
Ionic strength of pyrolytic gas at different heating rates (a) CH4(b) H2O(c) H2(d) CO2(e) CO.
Figure 4
TG / DTG curve of heating rate of coal sample at 10 °C/min
Figure 5
The relationship between ln[-ln(1-α)]/T2] and 1/T
Figure 6
Relationship between α and T at different heating rates (left)Relationship between ln(β/T2) and 1/T at
different conversion rates α(right)
Figure 7
Activation energy Ea versus different conversion rates α estimated from the Arrhenius plot.
