Effects of common mutations in the SARS-CoV-2 Spike RBD and its ligand, the human ACE2 receptor on binding affinity and kinetics
Since its identification in 2019 , a coronavirus able to induce a severe acute respiratory syndrome in humans , SARS-CoV-2 , has resulted in arguably the most severe infectious disease pandemic in 100 years . To date , more than 135 million people have been infected , resulting in the deaths from the resulting disease , COVID-19 , of more than 3 million people ( WHO , 2021 ) , and measures introduced to control spread have had harmful social and economic impacts . Fortunately , effective vaccines have been developed , and a global vaccination programme is underway ( Mahase , 2021 ) . New SARS-CoV-2 variants of concern are emerging that are making containment of the pandemic more difficult , perhaps by increasing transmissibility of the virus ( Davies et al . , 2021; Korber et al . , 2020; Volz et al . , 2021b; Volz et al . , 2021b; Washington et al . , 2021 ) and/or its resistance to protective immunity induced by previous infection or vaccines ( Darby and Hiscox , 2021; Dejnirattisai et al . , 2021; Garcia-Beltran et al . , 2021; Madhi et al . , 2021a; Madhi et al . , 2021b; Mahase , 2021; Volz et al . , 2021b; Volz et al . , 2021b ) . The SARS-CoV-2 virus enters cells following an interaction between the Spike ( S ) protein on its surface with angiotensin-converting enzyme 2 ( ACE2 ) on cell surfaces ( V’kovski et al . , 2021 ) . The receptor-binding domain ( RBD ) of the Spike protein binds the membrane-distal portion of the ACE2 protein . The S protein forms a homotrimer , which is cleaved shortly after synthesis into two fragments that remain associated non-covalently: S1 , which contains the RBD , and S2 , which mediates membrane fusion following the binding of Spike to ACE2 ( V’kovski et al . , 2021 ) . During the pandemic , mutations have appeared in the Spike protein that may increase transmissibility ( Davies et al . , 2021; Korber et al . , 2020; Richard et al . , 2021; Volz et al . , 2021b; Volz et al . , 2021b; Washington et al . , 2021 ) . One that emerged early in Europe , D614G , and quickly became dominant globally ( Korber et al . , 2020 ) , increases the density of intact Spike trimer on the virus surface by preventing premature dissociation of S1 from S2 following cleavage ( Zhang et al . , 2021; Zhang et al . , 2020 ) . A later mutation , N501Y , which has appeared in multiple lineages , lies within the RBD , and increases its affinity for ACE2 ( Starr et al . , 2020; Supasa et al . , 2021 ) . These findings suggest that mutations that directly or indirectly enhance Spike binding to ACE2 may increase transmissibility . Prior infection by SARS-CoV-2 and current vaccines induce antibody responses to the Spike protein , and most neutralising antibodies appear to bind to the Spike RBD ( Garcia-Beltran et al . , 2021; Greaney et al . , 2021a; Rogers et al . , 2020 ) . Some variants of concern have mutations in their RBD that confer resistance to neutralising antibodies ( Darby and Hiscox , 2021; Dejnirattisai et al . , 2021; Garcia-Beltran et al . , 2021; Madhi et al . , 2021a; Madhi et al . , 2021b; Mahase , 2021 ) . What is less clear is the precise effect of these mutations on the affinity and kinetics of the binding of RBD to ACE2 . Previous studies of the interaction between the Spike RBD and ACE2 have produced a wide range of affinity and kinetic estimates under conditions ( e . g . temperature ) that are not always well defined ( Lei et al . , 2020; Shang et al . , 2020; Supasa et al . , 2021; Wrapp et al . , 2020; Zhang et al . , 2021; Zhang et al . , 2020 ) . Precise information is needed to assess the extent to which RBD mutations have been selected because they enhance ACE2 binding or facilitate immune evasion . In this study , we undertook a detailed affinity and kinetic analysis of the interaction between Spike RBD and ACE2 at a physiological temperature ( 37°C ) , taking care to avoid common pitfalls . We used this optimised approach to analyse the effect of important common mutations identified in variants of RBD and ACE2 . Both mutations of ACE2 ( S19P , K26R ) and most of the mutations of RBD ( N501Y , E484K , and S477N ) enhanced the interaction , with one RBD mutation ( N501Y ) increasing the affinity by ~10-fold . Increased binding was the result of decreases in dissociation rate constants ( N501Y , S477N ) and/or increases in association rate constants ( N501Y , E484K ) . Although the K417N/T mutations found in the South African ( B . 1 . 351 ) and Brazilian ( P . 1 ) variants both decreased the affinity , the affinity-enhancing N501Y and E484K mutations that are also present in both variants confer a net ~4-fold increase in the affinity of their RBDs for ACE2 .
The focus of this study was to analyse common and therefore important variants of RBD and ACE2 . Henceforth , we will refer to the common ACE2 allele and RBD of the original SARS-CoV-2 strain sequenced in Wuhan as wild type ( WT ) . We chose mutations of RBD within the ACE2 binding site that have appeared independently in multiple SARS-CoV-2 lineages/clades ( Figure 1 , Figure 1—figure supplement 1; Hodcroft , 2021; Rambaut et al . , 2020 ) , suggesting that they confer a selective advantage , rather than emerged by chance , such as through a founder effect . The N501Y mutation has appeared in the Alpha ( B . 1 . 1 . 7; 20I/501Y . V1 ) , Beta ( B . 1 . 351; 20 H/501Y . V2 ) , and Gamma ( P . 1; 20 J/501Y . V3 ) variants , which were first identified in the UK , South Africa , and Brazil , respectively . The E484K mutation is present in the Beta and Gamma variants and has appeared independently in many other lineages , including Zeta ( P . 2; 20B/S . 484K ) , B . 1 . 1 . 318 , Eta ( B . 1 . 525; 20A/S:484 K ) , and Iota ( B . 1 . 526; 20 C/S . 484K ) . E484K has also appeared in VOC-202102–02 , a subset of the Alpha variant identified in the UK ( SARS-CoV-2 Variants of concern and variants under investigation - GOV . UK , 2021 ) . The S477N mutation became dominant for periods in Australia ( clade 20 F ) and parts of Europe ( 20A . EU2 ) and then appeared in New York in the Iota or B . 1 . 526 lineage ( Zhou et al . , 2021a ) . Mutations of K417 have appeared independently in the Beta and Gamma variants . Interestingly , N501Y , E484K , and S477N were the main mutations that appeared following random RBD mutagenesis and in vitro selection of mutants with enhanced ACE2 binding ( Zahradník et al . , 2021 ) . We selected for analysis the two most common mutations of ACE2 within the RBD binding site , K26R and S19P ( Figure 1C ) . They are present in 0 . 4% and 0 . 03% , respectively , of all samples in the gnomAD database ( Karczewski et al . , 2020 ) , while other ACE2 mutations in the RBD binding site are much less frequent ( <0 . 004% ) ( MacGowan et al . , 2021 ) . K26R is observed in all the major gnomAD populations but is most common in Ashkenazi Jews ( 1% ) and ( non-Finnish ) north-western Europeans ( 0 . 6% ) . It is less common in Africans/African-Americans and South Asians ( 0 . 1% ) and rare in Finnish ( 0 . 05% ) and East-Asian ( 0 . 001% ) populations . The S19P mutant is almost exclusively found in Africans/African-Americans ( 0 . 3% ) . To measure the effects of these mutations on the affinity and kinetics of the RBD/ACE2 interaction , we used surface plasmon resonance ( SPR ) , which allows very accurate measurements , provided that common pitfalls are avoided , particularly protein aggregation , mass-transport limitations , and rebinding ( van der Merwe and Barclay , 1996; Myszka , 1997 ) . Monomeric , soluble forms of the ectodomain of ACE2 and the Spike RBD were expressed in human cells , to retain native glycosylation , and purified ( Figure 2—figure supplement 1 ) . ACE2 was captured onto the sensor surface via a carboxy-terminal biotin and RBD injected over ACE2 at different concentrations ( Figure 2A ) . Excellent fits of 1:1 Langmuir binding model to the data yielded an association rate constant ( kon ) of 0 . 9 ± 0 . 05 μM–1s–1 and a dissociation rate constant ( koff ) of 0 . 067 ± 0 . 0011 s–1 ( mean ± SD , n = 6 , Table 1 ) . These rate constants are up to 25- fold faster than previously reported for the same interaction ( Lei et al . , 2020; Shang et al . , 2020; Supasa et al . , 2021; Wrapp et al . , 2020; Zhang et al . , 2021 ) . However , previous experiments were conducted at unphysiologically low temperatures ( i . e . below 37°C ) and under conditions in which mass-transport limitations and rebinding are highly likely ( see Discussion ) . These factors , and the presence of protein aggregates ( van der Merwe and Barclay , 1996 ) , would all lower the measured rate constants . In contrast , our measurements were conducted at 37°C and under conditions in which mass-transfer limitation and rebinding were excluded . The latter is demonstrated by the fact that measured kon and koff rates approached maximal values at the low level of ACE2 immobilisation ( ~50 RU ) used in our experiments ( Figure 2B , C; Dejnirattisai et al . , 2021 ) . The excellent fit of the 1:1 binding model to our data excludes an effect of protein aggregates , which yield complex kinetics . The calculated dissociation constant ( KD ) was 74 ± 4 nM ( mean ± SD , n = 6 , Table 1 ) . We also measured KD by equilibrium binding ( Figure 2D ) , which avoids any artefacts induced by mass transfer limitations and rebinding . This KD was very similar to the value calculated from kinetic data ( 63 ± 7 . 7 nM [mean ± SD , n = 24 , Table 1] ) , and did not vary with immobilisation level ( Figure 2E ) , further validating our kinetic measurements . These affinity values are within the wide range reported in previous studies , which varied from KD 6–133 nM ( Laffeber et al . , 2021; Lei et al . , 2020; Liu et al . , 2021; Shang et al . , 2020; Supasa et al . , 2021; Wrapp et al . , 2020; Zhang et al . , 2021 ) . We next evaluated the effect of RBD mutations on the affinity and kinetics of binding to ACE2 ( Figure 3 and Table 1 ) . Example sensorgrams are shown of mutations that increased ( N501Y , Figure 3A ) or decreased ( K417N , Figure 3B ) the binding affinity , while the key results from all mutants are summarised in Figure 3C . The single mutations S477N , E484K , and N501Y all enhanced binding . The N501Y mutation had the biggest effect , increasing the affinity ~10 fold to KD ~7 nM , by increasing the kon ~1 . 8-fold and decreasing the koff by ~7-fold . The S477N and E484K mutations increased the affinity more modestly ( ~1 . 5-fold ) , by decreasing the koff ( S477N ) or increasing the kon ( E484K ) . The K417T and K417N mutations decreased the affinity ~2- and ~ 4-fold , respectively , mainly by decreasing the kon but also by increasing the koff . Affinity-altering mutations in binding sites mainly affect the koff ( Agius et al . , 2013 ) and have more modest effects on the kon . Changes in electrostatic interactions can dramatically affect the kon ( Schreiber and Fersht , 1996 ) and are a plausible explanation for the effects of the mutations K417T , K417N , and E484K on kon . K417 forms a salt bridge with D30 on ACE2 ( Lan et al . , 2020 ) , while E484 is ~9 Å from E75 on ACE2 ( Lan et al . , 2020 ) . Thus , the mutations K417N/T and E484K would decrease and increase , respectively , long-range electrostatic forces that may accelerate association ( Schreiber and Fersht , 1996 ) . We also examined the effect on ACE2 binding of combinations of RBD mutations , including combinations present in VOC-202102–02 , a subset of the Alpha lineage ( N501Y ) with the E484K mutation ( “SARS-CoV-2 Variants of concern and variants under investigation – GOV . UK , ” 2021 ) , and the Beta and Gamma variants ( Figure 3C , Table 1 ) . In the case of VOC-202102–02 , the addition of the E484K mutation to N501Y further increased the affinity , to ~15-fold higher than WT RBD ( KD ~5 nM ) , by further increasing the kon . Because the higher kon could result in mass transfer limiting binding , we confirmed that the kinetic measurement for this variant was not substantially affected by varying levels of immobilisation ( Figure 3—figure supplement 1 ) . The affinity of the Beta ( K417N/ E484K/N501Y ) and Gamma ( K417T/E484K/N501Y ) RBD variants for ACE2 increased by 3 . 7- and 5 . 3-fold , respectively , relative to wild-type RBD , by both increasing the kon and decreasing the koff rate constants . We next examined whether the effects of the mutations were additive , as is typically the case for multiple mutations at protein/protein interfaces ( Wells , 1990 ) . To do this , we converted the changes in KD to changes in binding energy ( ΔΔG , Table 2 ) and examined whether the ΔΔG measured for RBD variants with multiple mutations was equal to the sum of the ΔΔG values measured for the individual RBD mutants . This was indeed the case ( Figure 3D ) , indicating that the effects on each mutation are independent . This is consistent with them being spaced well apart within the interface ( Figure 1C ) and validates the accuracy of the affinity measurements . We next examined the effects of mutations of ACE2 ( S19P and K26R ) on binding to both wild-type and common variants of RBD ( Figure 4 , Figure 4—figure supplement 1 , and Table 1 ) . Both S19P and K26R increased the affinity of WT RBD binding by ~3 . 7- and ~ 2 . 4-fold ( Figure 4A ) . These increases in affinity were the result of both increases in the kon and decreases in the koff . Finally , we looked for interactions between RBD and ACE2 mutations by measuring the effects of the ACE2 mutations on binding to all mutant forms of RBD ( Table 1 ) . After converting changes in KD to ΔΔG ( Table 2 ) , we examined whether ΔΔG measured for a given ACE2 variant/RBD variant interaction was equal to the sum of the ΔΔG measured for ACE2 variant/RBD WT and ACE WT/RBD variant interactions . This is depicted as the difference between the measured and predicted ΔΔG for interactions between ACE2 and RBD variants ( ΔΔΔG in Figure 4B , C , Dejnirattisai et al . , 2021 ) . In most cases , ΔΔΔG values were close to zero , indicating that the effects of these mutations were largely independent . The one exception was the combination of ACE2 S19P and RBD S477N variants , where the measured value was significantly lower than the predicted value ( Figure 4B ) , indicating that these mutations were not independent . This is consistent with the fact that the ACE2 residue S19 is adjacent to RBD residue S477 in the contact interface ( Figure 1C ) . An important consequence of this is that the S477N mutation increased the affinity of RBD for ACE2 WT but decreased its affinity for ACE2 S19P .
While our finding that the SARS-CoV-2 RBD binds ACE2 with an affinity of KD 74 nM at 37°C is consistent with previous studies ( KD 6–133 nM ) ( Laffeber et al . , 2021; Lei et al . , 2020; Liu et al . , 2021; Shang et al . , 2020; Supasa et al . , 2021; Wrapp et al . , 2020; Zhang et al . , 2021; Zhang et al . , 2020 ) , the rate constants that we measured ( kon 0 . 9 μM–1 . s–1 and koff 0 . 067 s–1 ) were faster than all previous reports . One likely reason for this is that previous measurements were performed at a lower temperature , which almost always decreases rate constants . While some studies stated that binding constants were measured at 25°C ( Laffeber et al . , 2021; Zhang et al . , 2020 ) , most studies did not report the temperature , suggesting that they were performed at room temperature or the standard instrument temperature ( 20°C–25°C ) . A second likely reason is that previous kinetic studies were performed under conditions in which the rate of diffusion of soluble molecule to the sensor surface limits the association rate , and rebinding of dissociated molecules to the surface reduces the measured dissociation rate . These are known pitfalls of both techniques used in these studies , surface plasmon resonance ( Myszka , 1997 ) , and bilayer interferometry ( Abdiche et al . , 2008 ) . In the present study , we avoided these issues by immobilising a very low level of ligand on the sensor surface . A third possible reason is that the proteins were aggregated , which can cause problems even when aggregates are a very minor contaminant ( van der Merwe and Barclay , 1996 ) . The presence of aggregates results in complex binding kinetics , which can be excluded if the simple 1:1 Langmuir binding model fits the kinetic data . While this was demonstrated in the present study , and some previous studies ( Shang et al . , 2020; Wrapp et al . , 2020; Zhang et al . , 2021 ) , such fits were not shown in all studies , one of which reported more than 20-fold slower kinetics than reported here ( Lei et al . , 2020; Supasa et al . , 2021 ) . The RBD mutations that we selected for analysis have all emerged independently and became dominant in a region at least once in different lineages , suggesting that they provide a selective advantage . Our finding that the N501Y , E484K , and S477N all increase the binding affinity of RBD for ACE2 raises the question as to whether this contributed to their selection . Several lines of evidence suggest that enhancing the Spike/ACE2 interaction would be advantageous for the virus . First , the virus has spread only very recently to humans from another mammalian host , providing insufficient time for optimisation of the affinity . Second , epidemiological studies have suggested that the Alpha variant , which has the N501Y mutation , has enhanced transmissibility ( Volz et al . , 2021a; Washington et al . , 2021 ) . Finally , a SARS-CoV-2 variant with the Spike mutation D614G , which increases its activity by stabilising it following furin cleavage ( Zhang et al . , 2021; Zhang et al . , 2020 ) , rapidly became dominant globally after it emerged ( Korber et al . , 2020; Volz et al . , 2021b ) . Taken together , these findings suggest that the WT Spike/ACE2 interaction is limiting for transmission and that mutations that enhance it , including the N501Y , E484K , and S477N mutations , could provide a selective advantage by increasing transmissibility . This raises two questions . First , will other RBD mutations appear in SARS-CoV-2 which further enhance transmission ? This seems likely , given that a large number of RBD mutations have been identified that increase the RBD/ACE2 affinity ( Starr et al . , 2020; Zahradník et al . , 2021 ) . Second , will combinations of existing mutations be selected because they further increase the affinity ? While the appearance E484K , together with N501Y in three lineages ( Alpha , Beta , and Gamma ) , supports this , it is also possible that E484K was selected because it disrupts antibody neutraliaation , as discussed below . Our affinity and kinetic data on RBD variants are broadly consistent with some ( Laffeber et al . , 2021; Liu et al . , 2021 ) , but not all ( Dejnirattisai et al . , 2021; Zhou et al . , 2021a ) , recent reports on the K417T/N , N501Y , and E484K variants . One caveat to our study is that we used monomeric forms of RBD and ACE2 . The native Spike protein is a trimer and has several other domains , including the nearby N-teminal domain , and the native ACE2 protein can exist as a dimer ( Yan et al . , 2020 ) . Because of these differences , our analysis may not detect the full effects of RBD and ACE2 mutations on the Spike/ACE2 interaction . A second caveat is that we have not examined the effect of these mutations on viral attachment to cells . Studies of other enveloped viruses , including SARS-CoV , suggest that increases in affinity of viral ligands for their cellular receptors can increase cell infection and disease severity ( Hasegawa et al . , 2007; Li et al . , 2005 ) . One study found that increasing this affinity enabled the virus to infect cells with lower receptor surface density ( Hasegawa et al . , 2007 ) . It follows that increases in affinity could increase the number of host tissues infected , which could increase the severity of disease ( Cao and Li , 2020 ) and/or increase the viral load in the upper respiratory tract ( Hoffmann et al . , 2020; Wölfel et al . , 2020 ) , thereby increasing spread . Another mechanism by which mutations of RBD could provide a selective advantage is through evasion of immune responses . This is supported by the observation that neutralising antibodies present in those infected by or vaccinated against SARS-CoV-2 primarily target the RBD ( Garcia-Beltran et al . , 2021; Greaney et al . , 2021a; Rogers et al . , 2020 ) . Furthermore , two variants with RBD mutations that abrogate antibody neutralisation , Beta and Gamma , became dominant in regions with very high levels of prior SARS-CoV-2 infection ( Cele et al . , 2021; Dejnirattisai et al . , 2021; Hoffmann et al . , 2021; Sabino et al . , 2021; Tegally et al . , 2021; Zhou et al . , 2021b ) . Both lineages include the N501Y mutation , but this appears to have modest effects on antibody neutralisation ( Greaney et al . , 2021a; Greaney et al . , 2021b ) . In contrast , the E484K mutation , also present in both variants , potently disrupts antibody neutralisation ( Greaney et al . , 2021a; Greaney et al . , 2021b ) . Our finding that the K417N/T mutations present in Beta and Gamma variants decrease the affinity of RBD for ACE2 suggests that they were selected because they facilitate immune escape . Indeed , mutations of K417 can block antibody neutralisation , albeit less effectively than E484K ( Greaney et al . , 2021a; Greaney et al . , 2021b; Wang et al . , 2021 ) . It is notable that these affinity-reducing K417N/T mutations have only emerged together with mutations ( N501Y and E484K ) that increase the affinity of RBD for ACE2 , suggesting a cooperative effect between mutations that enhance immune escape and mutations that increase affinity . The effect of the increased affinity for SARS-CoV-2 Spike RBD of the K26R and S19P ACE2 mutants is less clear . The evidence summarised above that WT RBD/ACE2 binding is limiting for SARS-CoV-2 transmission , suggest that carriers of these ACE2 variants will be at greater risk of infection and/or severe disease . However , in contrast to SARS-CoV-2 RBD mutations , the effects of ACE2 variants are primarily relevant to the carriers of these mutations . A preliminary analysis ( MacGowan et al . , 2021 ) suggests that the carriers of the K26R ACE allele might be at increased risk of severe disease , but the findings did not reach statistical significance , and further studies are required . The interaction that we identified between the RBD S477N and ACE2 S19P mutants highlights the importance of considering variation in the host population when studying the evolution of viral variants . In this case , the opposite effect of the RBD S477N mutation on its affinity for ACE2 S19P ( decreased ) , compared with ACE2 WT ( increased ) , suggests that this RBD variant may have a selective disadvantage amongst carriers of the ACE2 S19P variant , in contrast to those with ACE2 WT , where it appears to be advantageous . However , the low frequency of this variant means that this is unlikely to be important at a population level and will be difficult to detect . It is noteworthy that the two most common ACE2 variants are in positions on ACE2 with no known functional activity . This raises the question as to whether these mutations are a remnant of historic adaption to pathogens that utilised this portion of ACE2 . The fact that ACE2 S19P mutation is largely confined to African/African-American populations , suggests that it is more recent than K26R and/or selected by pathogen ( s ) confined to the African continent .
The plasmid used to express soluble ACE2 WT ( pOPINTTGneo_ACE2-BAP ) , which was kindly provided by Ray Owens ( Oxford Protein Production Facility-UK ) , encoded the following protein: The carboxy-terminal end has a biotin acceptor peptide ( underlined ) followed by an oligohistidine tag . The pCAGG plasmid used to express the RBD WT construct ( Amanat et al . , 2020 ) encoded the following protein: The carboxy-terminal end has an oligohistidine tag . ACE2 and RBD point mutations were introduced into these plasmid constructs using the Agilent QuikChange II XL Site-Directed Mutagenesis Kit following the manufacturer’s instructions . The primers were designed using the Agilent QuikChange primer design web program . Cells were grown in FreeStyle 293 Expression Medium ( ThermoFisher Scientific , 12338018 ) in a 37°C incubator with 8% CO2 on a shaking platform at 130 rpm . Cells were passaged every 2–3 days with the suspension volume always kept below 33 . 3% of the total flask capacity . The cell density was kept between 0 . 5 and 2 million per ml . Before transfection cells were counted to check that cell viability was above 95% , and the density was adjusted to 1 . 0 million per ml . For 100 ml transfection , 100 µl FreeStyle MAX Reagent ( ThermoFisher Scientific , 16447100 ) was mixed with 2 ml Opti-MEM ( ThermoFisher Scientific , 51985034 ) for 5 min . During this incubation , 100 µg of expression plasmid was mixed with 2 ml Opti-MEM ( or in situ biotinylation of ACE2 90 µg of expression plasmid was mixed with 10 µg of expression plasmid encoding the BirA enzyme ) . The DNA was then mixed with the MAX Reagent and incubated for 25 min before being added to the cell culture . For ACE2 in situ biotinylation , biotin was added to the cell culture at a final concentration of 50 µM . The culture was left for 5 days for protein expression to take place . Cells were harvested by centrifugation and the supernatant collected and filtered through a 0 . 22 μm filter . Imidazole was added to a final concentration of 10 mM and PMSF added to a final concentration of 1 mM; 1 ml of Ni-NTA Agarose ( Qiagen; 30310 ) was added per 100 ml of supernatant and the mix was left on a rolling platform at 4°C overnight . The mix was poured through a gravity flow column to collect the Ni-NTA Agarose . The Ni-NTA Agarose was washed three times with 25 ml of wash buffer ( 50 mM NaH2PO4 , 300 mM NaCl , and 20 mM imidazole at pH 8 ) . The protein was eluted with elution buffer ( 50 mM NaH2PO4 , 300 mM NaCl , and 250 mM imidazole at pH 8 ) . The protein was concentrated , and buffer exchanged into size exclusion buffer ( 25 mM NaH2PO4 and 150 mM NaCl at pH 7 . 5 ) using a protein concentrator with a 10 , 000 molecular weight cut-off . The protein was concentrated down to less than 500 μl and loading onto a Superdex 200 10/300 GL ( Cytiva , 17-5175-01 ) size exclusion column ( Figure 2—figure supplement 1 ) . Fractions corresponding to the desired peak were pooled and frozen at –80°C . Samples from all observed peaks were analysed on a reducing SDS–PAGE gel ( Figure 2—figure supplement 1 ) . RBD binding to ACE2 was analysed on a Biacore T200 instrument ( Cytiva ) at 37°C and a flow rate of 30 µl/min . Running buffer was HBS-EP ( Cytiva , BR100669 ) . Streptavidin was coupled with a CM5 sensor chip ( Cytiva , 29149603 ) using an amine coupling kit ( Cytiva , BR100050 ) to near saturation , typically 10 , 000–12 , 000 response units ( RU ) . Biotinylated ACE2 WT and variants were injected into the experimental flow cells ( FC2–FC4 ) for different lengths of time to produce desired immobilisation levels ( 20–800 RU ) . FC1 was used as a reference and contained streptavidin only . Excess streptavidin was blocked with two 40 s injections of 250 µM biotin ( Avidity ) . Before RBD injections , the chip surface was conditioned with eight injections of the running buffer . A dilution series of RBD was then injected in all FCs . Buffer alone was injected after every two or three RBD injections . The length of all injections was 30 s , and dissociation was monitored for 180–670 s . The background response measured in FC1 was subtracted from the response in the other three FCs . In addition , the responses measured during buffer injections closest in time were subtracted . Such double-referencing improves data quality when binding responses are low as needed to obtain accurate kinetic data ( Myszka , 1999 ) . At the end of each experiment , an ACE2-specific mouse monoclonal antibody ( NOVUS Biologicals , AC384 ) was injected at 5 µg/ml for 10 min to confirm the presence and relative amounts of immobilised ACE2 . Double-referenced binding data was fitted using GraphPad Prism . The koff was determined by fitting a mono-exponential decay curve to data from the dissociation phase of each injection . The koff from four to six RBD injections was averaged ( Figure 2—figure supplement 2A ) . The kon was determined by first fitting a mono-exponential association curve to data from the association phase , yielding the kobs , and then plotting the kobs vs the concentration of RBD and performing a linear fit of the equation kobs = kon*[RBD]+ koff to this data ( Figure 2—figure supplement 2B ) , using the koff determined as above to constrain the fit . The KD was either calculated ( calculated KD = koff/kon ) or measured directly ( equilibrium KD ) as follows . Equilibrium binding levels at a given [RBD] were determined from the fit of the mono-exponential association phase model to the association phase data . These equilibrium binding levels were plotted against [RBD] , and a fit of the simple 1:1 Langmuir binding model to this data was used to determine the equilibrium KD ( Figure 2D ) . ΔG for each affinity measurement was calculated from the relationship ΔG = R*T*lnKD , where R = 1 . 987 cal mol–1 K–1 , T = 310 . 18 K , and KD is in units M . ΔΔG values ( Table 2 and Figure 3D ) were calculated for each mutant from the relationship ΔΔG = ΔGWT – DGM . The predicted ΔΔG for interactions with multiple mutants were calculated by adding the single mutant ΔΔG values ( Figure 3D ) . The difference between the measured and predicted ΔΔG ( ΔΔΔG ) for interactions between the ACE2 and RBD mutants was calculates as ΔΔΔG = measured ΔΔG – predicted ΔΔG ( Figure 4B ) . All errors represent standard deviations and errors for calculated values were determined by error propagation .
The interaction between the SARS-CoV-2 virus Spike protein receptor binding domain ( RBD ) and the ACE2 cell surface protein is required for viral infection of cells . Mutations in the RBD are present in SARS-CoV-2 variants of concern that have emerged independently worldwide . For example , the B . 1 . 1 . 7 lineage has a mutation ( N501Y ) in its Spike RBD that enhances binding to ACE2 . There are also ACE2 alleles in humans with mutations in the RBD binding site . Here we perform a detailed affinity and kinetics analysis of the effect of five common RBD mutations ( K417N , K417T , N501Y , E484K , and S477N ) and two common ACE2 mutations ( S19P and K26R ) on the RBD/ACE2 interaction . We analysed the effects of individual RBD mutations and combinations found in new SARS-CoV-2 Alpha ( B . 1 . 1 . 7 ) , Beta ( B . 1 . 351 ) , and Gamma ( P1 ) variants . Most of these mutations increased the affinity of the RBD/ACE2 interaction . The exceptions were mutations K417N/T , which decreased the affinity . Taken together with other studies , our results suggest that the N501Y and S477N mutations enhance transmission primarily by enhancing binding , the K417N/T mutations facilitate immune escape , and the E484K mutation enhances binding and immune escape .
As the COVID-19 pandemic has progressed , new variants of the virus SARS-CoV-2 have emerged that are more infectious than the original form . The variants known as Alpha , Beta and Gamma have mutations in a protein on the virus’s surface that is vital for attaching to cells and infecting them . This protein , called Spike , carries out its role by binding to ACE2 , a protein on the surface of human cells . Mutations on Spike are found on the region where it binds to ACE2 . The interaction between these two proteins appears to be important to the behaviour of SARS-CoV-2 , but the impact of individual mutations in Spike is unknown . In addition , some people have different variants of ACE2 with mutations in the region that interacts with Spike , but it is not known whether this affects these people’s risk of contracting COVID-19 . To answer these questions , Barton et al . measured the precise effect of mutations in Spike and ACE2 on the strength of the interaction between the two proteins . The experiments showed that three of the five common Spike mutations in the Alpha , Beta and Gamma SARS-CoV-2 variants strengthened binding to ACE2 . The two mutations that weakened binding were only found together with other mutations that strengthened binding . This meant that the Spike proteins in all three of these SARS-CoV-2 variants bind to ACE2 more strongly than the original form . The experiments also showed that two common variants of ACE2 also increased the strength of binding to Spike . Interestingly , one of these ACE2 variants reversed the effect of a specific SARS-CoV-2 mutation , suggesting that carriers would be resistant to SARS-CoV-2 variants with this mutation . Identifying the precise effects of Spike mutations on ACE2 binding helps understand why new variants of SARS-CoV-2 spread more rapidly . This could help to identify concerning new variants before they spread widely and inform the response by health authorities . The finding that two common ACE2 variants bind more strongly to Spike suggests that people with these mutations could be more susceptible to SARS-CoV-2 .