Effects of common mutations in the SARS-CoV-2 Spike RBD and its ligand, the human ACE2 receptor on binding affinity and kinetics
- Sir William Dunn School of Pathology, University of Oxford, United Kingdom
- School of Life Sciences, University of Dundee, United Kingdom
Figures
Figure 1 with 1 supplement
Spike RBD and ACE2 variants analysed in this study.
(A) Phylogenetic tree illustrating the clades containing the RBD mutations investigated in this study. Constructed using TreeTime (Sagulenko et al., 2018) from the Nextstrain Global (Hadfield et al., 2018) sample of SARS-CoV-2 sequences from the GISAID database (Shu and McCauley, 2017) (accessed 15 April 2021, N = 4017). (B) Alignment illustrating the Spike residues that differ between SARS-CoV-2 variants, with the RBD mutants boxed. The variants are labelled with their clade designation from Nextstrain (Hadfield et al., 2018) and/or PANGO lineage (Rambaut et al., 2020), where relevant. The RBD mutations were collated from CoVariants (Hodcroft, 2021) and Nextstrain. (C) The structure of human ACE2 (green) in complex with SARS-CoV-2 Spike RBD (cyan). The area enclosed by the box is shown enlarged on the right, with the residues mutated in this study labelled. Drawn using UCSF Chimera (Pettersen et al., 2004) using coordinates from PDB 6m0j (Lan et al., 2020).
Figure 1—figure supplement 1
Emergence of the same RBD mutations in multiple SAR2-CoV-2 clades.
The figure highlights the SARS-CoV-2 clades containing RBD mutations investigated in this study. The phylogenetic trees were constructed as in Figure 1A from SARS-CoV-2 sequences accessed on 22 April 2021 (N = 3914). (A) N501Y has emerged independently in the three clades 501Y.V1, 501Y.V2, and 501Y.V3. Mutation to T at this position has also occurred frequently. (B) E484K has also been observed independently of its main progenitor clades 501Y.V2 and 501Y.V3. E484Q and E484G have also been observed. (C) S477N has been observed beyond clades 20 F and 20A.EU2. Mutations to I and R have also been occasionally observed at this position. (D) Mutations of K417 to N and T have been observed almost exclusively in the 20 H.501Y.V2 and 20 J.501Y.V3 clades.
Figure 2 with 2 supplements
SPR analysis.
(A) Overlay of traces showing association and dissociation when WT RBD is injected for 30 s at the indicated concentration over immobilised WT ACE2. The right panel shows an expanded view of the dissociation phase. The blue lines show the fits used for determining the kon and koff. The kon was determined as described in Figure 2—figure supplement 2. The koff (B) and kon (C) values measured at different levels of immobilised ACE2 are shown. (D) The equilibrium KD was determined by plotting the binding at equilibrium against [RBD] injected. Data from experiment shown in (A). (E) The equilibrium KD measured at different levels of immobilised ACE2 are shown.
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Figure 2—source data 1
Source data for Figure 2.
- https://cdn.elifesciences.org/articles/70658/elife-70658-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Protein purification.
Size-exclusion chromatography traces of the indicated ACE2 and RBD proteins and reducing SDS–PAGE of the indicated peak fractions. UK2 refers to the VOC-202102–02 variants. In preparations of RBD, unidentified ~60 kDa contaminants were present at various levels, but always <5% by densitometry.
Figure 2—figure supplement 2
Determining the kon and koff.
Analysis of data from the fits in Figure 2A. (A) A plot of koff obtained for each injection versus [RBD]. (B) A plot of kobs for each injection versus [RBD]. The line shows a constrained fit of the equation kobs = kon*[RBD]+ koff, using the koff obtained in (A). The kon was obtained from the slope.
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Figure 2—figure supplement 2—source data 1
Source data for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/70658/elife-70658-fig2-figsupp2-data1-v3.xlsx
Figure 3 with 2 supplements
Effect of RBD mutations on binding to WT ACE2.
Overlay of traces showing association and dissociation of N501Y (A) and K417N (B) RBD variants when injected at a range of concentrations over immobilised WT ACE2. The right panels show an expanded view of the dissociation phase. The blue lines show fits used for determining the kon and koff. (C) The fold change relative to WT RBD of the calculated KD, kon, and koff for binding of the indicated RBD variants to immobilised WT ACE2 (error bars show SD, n = 3). Representative sensorgrams from all mutants shown in Figure 3—figure supplement 2, and the mean values from multiple repeats are in Table 1. (D) The blue lines show the measured ΔΔG for indicated RBD variants. The red lines show the predicted ΔΔG for the RBD variants with multiple mutations, which were calculated by adding ΔΔG values for single mutation variants (error bars show SD, n = 3).
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Figure 3—source data 1
Source data for Figure 3.
- https://cdn.elifesciences.org/articles/70658/elife-70658-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Mass transport controls for RBD.
The koff (A) and kon (B) for E484K/N501Y (UK2) RBD binding WT ACE2 at a range of surface immobilisations (n = 12). UK2 refers to VOC-202102–02.
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Figure 3—figure supplement 1—source data 1
Source data for Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/70658/elife-70658-fig3-figsupp1-data1-v3.xlsx
Figure 3—figure supplement 2
Representative SPR data for RBD variants binding to WT ACE2.
Binding traces for the indicated RBD variants injected at different concentrations over immobilised WT ACE2. The right panels show an expanded view of the dissociation phase. The blue lines show fits used for determining the kon and koff. UK2 refers to the VOC-202102–02 variant.
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Figure 3—figure supplement 2—source data 1
Source data for Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/70658/elife-70658-fig3-figsupp2-data1-v3.xlsx
Figure 4 with 1 supplement
Effect of mutations in ACE2.
(A) The fold change relative to WT ACE2 of the calculated KD, kon, and koff for the interaction of WT RBD and the indicated ACE2 variants (error bars show SD, n = 3). (B, C) Show the difference (ΔΔΔG) between the measured and predicted ΔΔG for S19P (B) and K26R (C) ACE2 variants binding to the indicated RBD variants, calculated from data in Table 2. The predicted ΔΔG values for each variant RBD/variant ACE2 interaction were calculated from the sum of the ΔΔG for the ACE2 variant binding WT RBD and the ΔΔG for the RBD variant binding WT ACE2 (Table 2).
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Figure 4—source data 1
Source data for Figure 4.
- https://cdn.elifesciences.org/articles/70658/elife-70658-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Representative SPR data for WT RBD binding ACE2 variants.
Binding traces for the WT RBD injected at different concentrations over the indicated immobilised ACE2 variants. The right panels show an expanded view of the dissociation phase. The blue lines show fits used for determining the kon and koff.
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Figure 4—figure supplement 1—source data 1
Source data for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/70658/elife-70658-fig4-figsupp1-data1-v3.xlsx
Tables
Table 1
Affinity and kinetic data for RBD variants and ACE2 variants.
Mean and SD of the koff, kon, calculated KD, and equilibrium KD values for all RBD variants binding all ACE2 variants. For most measurements n = 3, the exceptions were RBD WT/ACE2 WT equilibrium KD measurements (n = 24) and other RBD WT measurements (n = 6). UK2 refers to the VOC-202102–02 variant.
| koff (s–1) | SD | kon (µM–1 s–1) | SD | KD calc. (nM) | SD | KD equi. (nM) | SD | |
|---|---|---|---|---|---|---|---|---|
| RBD over WT ACE2 | ||||||||
| WT | 0.0668 | 0.00113 | 0.90 | 0.05 | 74.4 | 4.0 | 62.6 | 7.7 |
| K417N | 0.177 | 0.00416 | 0.49 | 0.05 | 364 | 29 | 349 | 10 |
| K417T | 0.126 | 0.00510 | 0.55 | 0.04 | 230 | 23 | 226 | 19 |
| S477N | 0.0348 | 0.00037 | 0.81 | 0.03 | 42.9 | 2.1 | 42.6 | 3.0 |
| E484K | 0.0818 | 0.00183 | 1.54 | 0.03 | 53.1 | 1.7 | 52.6 | 2.0 |
| N501Y (Alpha) | 0.0111 | 0.00017 | 1.59 | 0.04 | 7.0 | 0.25 | 5.5 | 2.4 |
| K417N/E484K | 0.251 | 0.00799 | 1.02 | 0.07 | 247 | 23 | 251 | 23 |
| K417T/E484K | 0.168 | 0.00573 | 1.10 | 0.05 | 153 | 12 | 147 | 8.6 |
| E484K/N501Y (UK2) | 0.0118 | 0.00037 | 2.33 | 0.10 | 5.1 | 0.36 | 3.7 | 2.7 |
| K417N/E484K/N501Y (Beta) | 0.0291 | 0.00076 | 1.46 | 0.06 | 20.0 | 0.70 | 17.4 | 3.1 |
| K417T/E484K/N501Y (Gamma) | 0.0211 | 0.00021 | 1.56 | 0.07 | 13.5 | 0.45 | 12.2 | 3.4 |
| RBD over S19P ACE2 | ||||||||
| WT | 0.0298 | 0.00039 | 1.50 | 0.12 | 20.0 | 1.3 | 30.5 | 2.2 |
| K417N | 0.0782 | 0.00284 | 0.72 | 0.04 | 108 | 2.8 | 129 | 8.2 |
| K417T | 0.0521 | 0.00196 | 0.69 | 0.02 | 75.8 | 4.7 | 87.8 | 7.0 |
| S477N | 0.0257 | 0.00016 | 1.05 | 0.07 | 24.6 | 1.7 | 30.3 | 2.7 |
| E484K | 0.0325 | 0.00031 | 2.02 | 0.08 | 16.2 | 0.55 | 20.8 | 1.3 |
| N501Y (Alpha) | 0.0051 | 0.00004 | 2.31 | 0.09 | 2.2 | 0.09 | 3.5 | 0.4 |
| K417N/E484K | 0.0961 | 0.00198 | 1.28 | 0.11 | 75.6 | 7.1 | 91.3 | 6.5 |
| K417T/E484K | 0.0660 | 0.00255 | 1.45 | 0.03 | 45.5 | 2.5 | 53.8 | 1.5 |
| E484K/N501Y (UK2) | 0.0051 | 0.00008 | 3.10 | 0.10 | 1.7 | 0.05 | 3.4 | 0.4 |
| K417N/E484K/N501Y (Beta) | 0.0122 | 0.00009 | 2.16 | 0.03 | 5.7 | 0.07 | 10.4 | 1.2 |
| K417T/E484K/N501Y (Gamma) | 0.0085 | 0.00007 | 2.11 | 0.05 | 4.0 | 0.07 | 6.1 | 1.3 |
| RBD over K26R ACE2 | ||||||||
| S477N | 0.0240 | 0.00009 | 1.07 | 0.05 | 22.6 | 1.1 | 33.4 | 1.3 |
| WT | 0.0500 | 0.00062 | 1.60 | 0.16 | 31.4 | 2.6 | 48.8 | 2.5 |
| K417N | 0.154 | 0.00789 | 0.88 | 0.07 | 175 | 8.1 | 237 | 15 |
| K417T | 0.101 | 0.00079 | 0.81 | 0.12 | 127 | 17.4 | 154 | 2.8 |
| S477N | 0.0240 | 0.00009 | 1.07 | 0.05 | 22.6 | 1.1 | 33.4 | 1.3 |
| E484K | 0.0587 | 0.00109 | 2.03 | 0.03 | 28.9 | 1.0 | 35.9 | 1.5 |
| N501Y (Alpha) | 0.0081 | 0.00002 | 2.34 | 0.09 | 3.5 | 0.15 | 7.5 | 1.5 |
| K417N/E484K | 0.191 | 0.00481 | 1.48 | 0.15 | 130 | 9.4 | 166 | 11 |
| K417T/E484K | 0.135 | 0.00407 | 1.53 | 0.02 | 88.0 | 3.9 | 105 | 0.7 |
| E484K/N501Y (UK2) | 0.0085 | 0.00018 | 3.06 | 0.23 | 2.8 | 0.17 | 6.4 | 0.3 |
| K417N/E484K/N501Y (Beta) | 0.0234 | 0.00040 | 2.13 | 0.05 | 11.0 | 0.28 | 18.7 | 2.0 |
| K417T/E484K/N501Y (Gamma) | 0.0164 | 0.00028 | 2.21 | 0.06 | 7.4 | 0.33 | 15.3 | 0.8 |
Table 2
ΔΔG for RBD variants binding to ACE2 variants.
Mean and SD of ΔΔG (n = 3, kcal/mol) were determined as described in Materials and methods using the calculated KD values in Table 1. UK2 refers to the VOC-202102–02 variant.
| Ace2 wt | Ace2 s19p | Ace2 k26r | ||||
|---|---|---|---|---|---|---|
| RBD variant | ΔΔG | SD | ΔΔG | SD | ΔΔG | SD |
| WT | 0.00 | 0.00 | 0.79 | 0.05 | 0.52 | 0.06 |
| K417N | –0.96 | 0.06 | –0.23 | 0.04 | –0.52 | 0.04 |
| K417T | –0.68 | 0.07 | –0.01 | 0.05 | –0.32 | 0.09 |
| S477N | 0.33 | 0.04 | 0.67 | 0.05 | 0.72 | 0.04 |
| E484K | 0.20 | 0.04 | 0.92 | 0.04 | 0.57 | 0.04 |
| N501Y (Alpha) | 1.43 | 0.04 | 2.13 | 0.04 | 1.86 | 0.04 |
| K417N/E484K | –0.72 | 0.07 | –0.01 | 0.07 | –0.34 | 0.06 |
| K417T/E484K | –0.43 | 0.06 | 0.30 | 0.05 | –0.10 | 0.04 |
| E484K/N501Y (UK2) | 1.62 | 0.05 | 2.30 | 0.04 | 1.98 | 0.05 |
| K417N/E484K/N501Y (Beta) | 0.79 | 0.04 | 1.56 | 0.03 | 1.16 | 0.04 |
| K417T/E484K/N501Y (Gamma) | 1.03 | 0.04 | 1.76 | 0.03 | 1.39 | 0.04 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Transfected construct (human) | ACE2 WT | Oxford Protein Production Facility-UK | pOPINTTGneo_ACE2-BAP | T |
| Transfected construct (human) | ACE2 S19P; ACE2 K26R | This paper | Available from authors | |
| Transfected construct (SARS-CoV-2) | RBD WT | BEI Resources, NIH | NR-52309 | pCAGG plasmid |
| Transfected construct (SARS-CoV-2) | RBD K417N; RBD RBD K417T; RBD S477N; RBD E484K; RBD N501Y; RBD K417N/E484K; RBD K417T/E484K; RBD beta; RBD gamma | This paper | pCAGG plasmid. Available from authors | |
| Transfected construct (human) | pTT3-BirA-FLAG | Addgene | RRID:Addgene_64395 | Cotranfected for in-cell biotinylation |
| Peptide, recombinant protein | ACE2 WT; ACE2 S19P; ACE2 K26R | This paper | Expressed in HEK293 cells and purified | |
| Peptide, recombinant protein | RBD WT; RBD K417N; RBD K417T; RBD S477N; RBD E484K; RBD N501Y; RBD K417N/E484K; RBD K417T/E484K; RBD beta; RBD gamma | This paper | Expressed in HEK293 cells and purified | |
| Antibody | anti-human ACE2 (mouse monoclonal) | NOVUS Biologicals | AC384 | (5 µg/mL) |
| Cell line (human) | FreeStyle HEK293F Cells | ThermoFisher Scientific | RRID:CVCL_D603 | |
| Chemical compound, drug | FreeStyle MAX Reagent | ThermoFisher | 16447100 | |
| Chemical compound, drug | FreeStyle 293 Expression Medium | ThermoFisher | 12338018 | |
| commercial assay or kit | QuikChange II XL | Agilent | 200,521 | |
| Commercial assay or kit | Amine coupling kit | Cytiva | BR100050 | |
| Software, algorithm | GraphPad | Prism | Version 9 | |
| Other | CM5 sensor chips | Cytiva | 29149603 |
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Effects of common mutations in the SARS-CoV-2 Spike RBD and its ligand, the human ACE2 receptor on binding affinity and kinetics
eLife 10:e70658.
https://doi.org/10.7554/eLife.70658
