(C) PLOS One
This story was originally published by PLOS One and is unaltered.
 . . . . . . . . . .



In vitro evolution predicts emerging SARS-CoV-2 mutations with high affinity for ACE2 and cross-species binding [1]

['Neil Bate', 'Department Of Molecular', 'Cell Biology', 'University Of Leicester', 'Leicester', 'Leicester United Kingdom', 'Department Of Cardiovascular Sciences', 'Christos G. Savva', 'Leicester Institute Of Structural', 'Chemical Biology']

Date: 2022-09 

Emerging SARS-CoV-2 variants are creating major challenges in the ongoing COVID-19 pandemic. Being able to predict mutations that could arise in SARS-CoV-2 leading to increased transmissibility or immune evasion would be extremely valuable in development of broad-acting therapeutics and vaccines, and prioritising viral monitoring and containment. Here we use in vitro evolution to seek mutations in SARS-CoV-2 receptor binding domain (RBD) that would substantially increase binding to ACE2. We find a double mutation, S477N and Q498H, that increases affinity of RBD for ACE2 by 6.5-fold. This affinity gain is largely driven by the Q498H mutation. We determine the structure of the mutant-RBD:ACE2 complex by cryo-electron microscopy to reveal the mechanism for increased affinity. Addition of Q498H to SARS-CoV-2 RBD variants is found to boost binding affinity of the variants for human ACE2 and confer a new ability to bind rat ACE2 with high affinity. Surprisingly however, in the presence of the common N501Y mutation, Q498H inhibits binding, due to a clash between H498 and Y501 side chains. To achieve an intermolecular bonding network, affinity gain and cross-species binding similar to Q498H alone, RBD variants with the N501Y mutation must acquire instead the related Q498R mutation. Thus, SARS-CoV-2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in emerging SARS-CoV-2 variants where they have the potential to influence human-to-human and cross-species transmission.

The ability to predict mutations in SARS-CoV-2 that cause increased infectivity or immune evasion would be valuable in development of vaccines and therapeutics, and prioritizing viral variants for monitoring and containment. Using rapid in vitro evolution, we identify mutations in SARS-CoV-2 receptor binding domain (RBD) that markedly increase binding of RBD to ACE2. One mutation in particular, Q498H, causes a substantial increase in binding affinity, and we uncover the mechanism for this by structural determination. We show addition of Q498H increases binding of some SARS-CoV-2 variant RBDs but find Q498H is incompatible with the common N501Y mutation. RBD variants with N501Y can instead access similar bonding and affinity increases by acquiring the alternative Q498R mutation. The Q498R/N501Y combination mutations, and Q498H, also enable de novo high affinity binding of SARS-CoV-2 RBD to rat ACE2. Our data show SARS-CoV-2 RBD can access large affinity gains and cross-species binding via two alternative mutational routes involving Q498, with route selection determined by whether a variant already has the N501Y mutation. These mutations are now appearing in new SARS-CoV-2 variants, where they have the potential to contribute to increased variant transmissibility and potentially facilitate cross-species transmission.

Funding: This work was funded by the Medical Research Council (MC_PC_19043). NB and NPJB are supported by the British Heart Foundation (PG/19/27/34305). PCEM is a Royal Society Wolfson Fellow (RSWF\R3\183003). Work in the JES group is supported by a core grant to the LMB from the MRC (U105178808). The Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB) received major funding from MRC (MC_PC_17136). The funders did not play any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Bate et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

We have been using a rapid and facile cell surface display evolution approach to seek new mutations and variants of spike protein RBD with functional effects of concern. With this approach we identify an RBD mutant containing S477N and Q498H mutations that exhibits an almost 7-fold increase in binding affinity for human ACE2. We solve the mutant RBD:ACE2 structure to reveal the binding mechanism. The affinity gain of the double mutant is mainly due to the Q498H mutation, and we show this mutation also boosts binding of other RBD variants. Surprisingly however, Q498H inhibits RBD binding if a SARS-CoV-2 variant already has the N501Y mutation, and we find this is due to a clash between the aromatic side chains. In contrast, the related Q498R mutation is compatible with N501Y, and it exhibits positive epistasis with Y501 to enhance binding to ACE2. Importantly, we find that Q498R plus N501Y, as well as Q498H, can also enable variants to bind rat ACE2 with high affinity, opening potential SARS-CoV-2 transmission routes for such variants between humans and rodents. Our data show Q498 to be a pivotal position by which SARS-CoV-2 can access large affinity gains and cross-species binding via two alternative mutational routes, with route selection determined by whether the variant already has the N501Y mutation. These mutations have now been found in new SARS-CoV-2 variants and they may contribute to increased variant transmissibility in the human population and potentially facilitate cross-species transmission.

The ability to predict the combinations of mutations that can arise in SARS-CoV-2, and understand how these affect transmissibility and other functions, is critical for development of broadly acting therapeutics and vaccines that will be effective as the virus evolves. This knowledge is also crucial for early identification of variants that should be prioritised for monitoring and containment. Deep mutational scanning of the receptor binding domain (RBD) of SARS-CoV-2 spike protein has provided valuable insights into potential effects of individual mutations affecting stability and affinity for ACE2 [ 9 ]. In vitro evolution is another powerful methodology that can be used to identify mutations that affect protein activities, and it is particularly effective for revealing combinations of mutations that work together to modify functions.

As the number of SARS-CoV-2 infected individuals rise, in human and other hosts, the opportunities for the appearance of viral mutations resulting in enhanced infectivity or pathogenesis increase. With rising immunity to the virus, there is growing selective pressure on the virus for immune evasion. Consequently, viral variants are emerging with spike protein mutations that enable antibody escape, such as the K417N mutation [ 3 ]. Some immune escape mutations lead to a decrease in binding affinity of spike protein for ACE2, and for such mutations, successful viral lineages are those that have combined the escape mutation with additional mutations that restore binding affinity. An example of this is the SARS-CoV-2 B.1.351 (Beta) variant that contains K417N and N501Y mutations. K417N suppresses binding of a number of antibodies [ 3 ], but also decreases binding affinity to ACE2 by around five-fold [ 7 , 8 ]. The addition of an N501Y mutation enhances binding affinity and can restore the decrease in affinity caused by K417N [ 7 , 8 ]. With selective pressure for immune escape, mutations that increase binding affinity of the spike protein become more important as they enable the virus to explore a wider range of escape mutations, including those that would otherwise hamper binding to ACE2.

In addition to modifying viral transmissibility and immune escape, mutations in the spike protein can also enable the virus to bind ACE2 in species not previously susceptible to infection. Extending the range of species that a virus can infect allows the emergence of new host species and can have important health implications by creating new viral reservoirs with the potential to re-infect the human population. For example, SARS-CoV-2 has been transmitted to mink and spilled back from mink to humans [ 6 ]. This potential for cross-species transmission is especially pertinent when viruses infect species that live in close proximity to humans. Importantly, viral evolution in these new reservoirs, and recombination with other coronaviruses, has the potential to create further novel variants, that could potentially cross back into humans.

SARS-CoV-2 emerged in late 2019 and has led to more than 420 million cases of COVID-19 infection worldwide as of February 2022 [ 1 ]. The disease is caused by the betacoronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and arose by zoonotic transmission of the virus from an animal reservoir, most likely horseshoe bat (Rhinolophus) via an intermediate host [ 2 ]. Since entering the human population, the virus has continued to evolve and adapt to maximise its fitness. This is leading to the emergence of viral variants with increased transmissibility as well as the ability to evade therapeutics and antibodies induced by infection or vaccination [ 3 ]. Many of the mutations found in these variants are localised to the viral spike protein, which binds to the host cell receptor angiotensin converting enzyme-2 (ACE2) enabling viral infection [ 4 , 5 ].

Results

The DT40 cell surface display system [10,11] was used to identify potential spike protein variants with increased affinity for ACE2. To do this DT40 cells expressing SARS-CoV-2 RBD (residues 19–541) on their surface (Fig 1a) were incubated with the indicated concentrations of monomeric ACE2 (residues 19–615, with a HA epitope tag and C-terminal Histidine 6 ) and bound ACE2 detected with anti-Histidine 6 , along with RBD expression level detected using anti-FLAG. Pools of cells with the highest ACE2 binding were selected by taking diagonal sort windows to normalize for RBD expression (Fig 1b). The number of rounds of selection was limited to three to favour selection of variants with a minimal number of accessible mutations, and sort windows were set to capture variants with maximum affinity gain. DNA encoding RBD was amplified by PCR from genomic DNA of the selected cells and sequenced. The dominant variant recovered had the double mutations S477N and Q498H and was designated S477N/Q498H-RBD (Fig 1c). These mutations are located at each end of the binding interface with ACE2 (Fig 1d).

PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 1. Selection of SARS-CoV-2 receptor binding domain with high affinity for HuACE2. (a) Schematic representation of cell surface expressed SARS-CoV-2 RBD showing RBD, FLAG-epitope tag and PDGF-receptor transmembrane anchor. (b) SARS-CoV-2 RBD was selected for enhanced ACE2 binding by somatic hypermutation and cell surface display in DT40 cells. FACS plots are shown of DT40 cells following binding of Histidine 6 -tagged-HuACE2. Bound ACE2 was detected with anti-Histidine 6 -phycoerythrin (anti-His 6 -PE) and expression level of RBD was assessed with ant-FLAG-allophycocyanin (anti-FLAG-APC). Sort windows are indicated for each round of selection and diversification, selecting at each round for highest Ang2 binding. Selections were performed at 0.1nM ACE2. The final panel shows a binding plot of the selected cells. (c) Amino acid mutations S477N and Q498H in the selected RBD are shown, along with the corresponding single nucleotide mutations. Mutated residues and nucleotides are in red. (d) Position of residue mutations in the selected RBD. Mutated residues are shown in red on the ACE2 binding interface (PDB accession number 6M0J [13]). The RBD is orientated to show the face that interacts directly with ACE2. https://doi.org/10.1371/journal.ppat.1010733.g001

S477N and Q498H mutations have already been reported in isolates from patients. As of February 2022, S477N containing SARS-CoV-2 variants have been found in multiple countries and this mutation has emerged at least seven times during the pandemic [12]. The Q498H mutation is currently much rarer, with 47 genotyped cases being reported in the GISAID database as of February 2022, and around 1.4 million cases with the related Q498R mutation, most of these being in the Omicron variant which also has the S477N mutation [12]. Infections have occurred worldwide. The co-occurrence of S477N and Q498H has yet to be reported.

SARS-CoV-2 RBDs with Q498H mutations have high affinity for ACE2 The binding ability of the soluble epitope-tagged RBD of Wuhan-Hu-1 SARS-CoV-2 (WH-RBD) and mutant RBD (Fig 2a) was measured using biolayer interferometry (BLI) with human ACE2 (Hu-ACE2) immobilized on the biosensor (Fig 2b). In our experiments WH-RBD binds Hu-ACE2 with a K D of 9.3nM, whereas S477N/Q498H-RBD bound Hu-ACE2 with a K D of 1.4nM, representing around 6.5-fold higher affinity of binding than WH-RBD (Fig 2b and 2c). To assess the contribution of each mutation to the overall increase in affinity, we assayed the Q498H and S477N mutations individually. RBD with Q498H bound ACE2 with a K D of 2.9nM. The S477N mutant was found to bind ACE2 with a K D of 4.0nM (Fig 2b and 2c). In S477N/Q498H-RBD, N477 and H498 are positioned at each end of the RBD binding interface and work together to stabilize the RBD:ACE2 complex, once formed, as indicated by the more than 4-fold decrease in K off observed for the combined mutant (Fig 2b and 2c). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 2. SARS-CoV-2 RBDs with Q498H mutations have high affinity for ACE2. (a) Soluble forms of HuACE2, WH-RBD and mutant RBD were expressed in HEK 293 cells and purified on nickel affinity columns. Purified proteins were resolved by SDS/PAGE and detected by Coomassie staining. Molecular masses are shown in kDa. (b) Biolayer interferometry plots of kinetics of binding RBD to HuACE2. WH-RBD, or the indicated RBD, was in solution and Hu-ACE2 immobilized on sensors. Binding was measured with RBD concentrations of 12, 30, 60, 120nM for WH-RBD and 6, 12, 30, 60nM for the mutants. Fitted curves are in red. Data are shown for single experiments representative of at least three independent experiments for each RBD. (c) Kinetic binding constants for RBD binding to HuACE2 measured using biolayer interferometry. Data are shown as means and SEM for at least three independent experiments. (d) Cellular binding of RBD and S477N/Q498H-RBD. Vero-E6 cells were incubated without RBD (C) or with a range of concentrations of WH-RBD (circles) or S477N/Q498H-RBD (triangles) as indicated, at 37°C for 15 mins before washing, antibody staining of bound RBD with fluorescently conjugated antibody, and flow cytometry. Data are shown as mean fluorescence intensity vs RBD concentration (nM) for a single experiment representative of three. https://doi.org/10.1371/journal.ppat.1010733.g002 To test whether the increase in binding of S477N/Q498H-RBD for ACE2 seen in BLI assays is reflected in an increase in binding affinity in the cellular context we used flow cytometry (Fig 2d). Vero-E6 cells, which express ACE2, were incubated with varying concentrations of WH-RBD or S477N/Q498H-RBD, cells were washed and bound RBD was detected with a fluorescent antibody recognizing the epitope tag on the RBD proteins. The mean fluorescence intensity of the cells was measured by flow cytometry and plotted against RBD concentration. Both WH-RBD and S477N/Q498H-RBD bound in a concentration dependent manner (Fig 2d). The concentration of WH-RBD required for half-maximal binding was 17.4 +/- 0.3nM compared with 4.3 +/- 0.4nM (mean and SEM for three independent experiments) for S477N/Q498H-RBD. Consistent with the data on in vitro binding to purified ACE2, therefore, S477N/Q498H-RBD bound with higher apparent affinity than WH-RBD.

Structure of S477N/Q498H-RBD in complex with HuACE2 The increase in affinity of S477N/Q498H mutated RBD, and particularly the substantial increase in binding due to the Q498H mutation, prompted us to determine the structure of the S477N/Q498H-RBD:ACE2 complex to gain insight into the binding mechanism. The complex was imaged by Cryo-EM to produce a good quality 3.2Å map for which we could build an atomic model for the complex and observe the new interactions across the binding interface (Fig 3a and 3b). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 3. Structure of S477N/Q498H-RBD in complex with HuACE2. Cryo-EM structure and model of the ACE2-RBD complex. Sharpened Cryo-EM map (a) of the HuACE2-S477N/Q498H-RBD complex with ACE2 coloured yellow and RBD in green. The refined coordinates shown in cartoon representation (b) and coloured as above. In panel (c) H498 can be seen in proximity to ACE2 Y41 forming a non-planar π-interaction while ACE2 residues K353 and D38 are within hydrogen bonding distance to H498 and could contribute to the tighter interaction formed by this RBD mutant, whereas Panel (d) shows Q498 in WH-RBD (PDB accession number 6M0J [13]) in proximity to ACE-2 Q42. Panel (e) indicates that the S477N mutation places this longer side-chain closer to S19 in ACE2 and within hydrogen bonding distance thus enhancing the binding between HuACE2 and S477N/Q498H-RBD. Panel (f) shows positioning of S477 in WH-RBD and ACE2 S19 (PDB accession number 6M0J [13]). https://doi.org/10.1371/journal.ppat.1010733.g003 Examination of the structure reveals mutation of RBD Q498 for histidine results in a perpendicular Y-shaped π-interaction between ACE2 Y41 and RBD H498 (Fig 3c). In addition, RBD H498 now lies within hydrogen bonding distance of a flexible ACE2 lysine residue (K353) which would strengthen the interaction in this area. A neighbouring aspartate side chain (ACE2 D38) could also mediate a hydrogen bond via a water molecule to RBD H498 and this is supported by a continuous density between ACE2 D38 and RBD H498 at lower contour levels. Although we are unable to build water molecules into the structure at this resolution, we do find several densities in our map coincide exactly with the water molecules built for the x-ray model used as starting coordinates for this study (PDB 6M0J; [13]). On the other side of the interface between ACE2 and RBD the gain of a carbon atom from the RBD S477N mutation places this side chain in hydrogen bonding distance of ACE2 S19 (Fig 3e), whereas S477 in the un-mutated RBD is unable to reach S19 (Fig 3f). In addition to the newly formed interactions, we examined how the new binding interface may be further favourable over the Wuhan-Hu-1 interface. Examination of the difference electron density map of PDB 6M0J [13] reveals that although RBD Q498 can hydrogen bond with ACE2 Q42 (Fig 3d), the positive difference electron density adjacent to the side chain is consistent with disorder (i.e. it adopts at least one alternate conformation) (S1 Fig). Alternate conformations would break the hydrogen bond with ACE2 Q42 suggesting that the contribution of RBD Q498 to ACE2 binding is reduced. This disorder in Q498 can be seen in all but one of the crystal structures in the PDB.

Q498H and Q498R mutations modify binding of B.1.617.1/3 and B.1.351 variant RBD to HuACE2 The finding that mutating histidine at position 498 in SARS-CoV-2 RBD causes a dramatic increase in binding affinity for ACE2 prompted us to test whether this mutation could enhance binding of other SARS-CoV-2 RBD variants. We therefore added the Q498H mutation to two prominent RBD variants, one containing the mutations L452R and E484Q, which are common to variants B.1.617.1 (Kappa) and B.1.617.3 (Delta), and one containing RBD mutations K417N, E484K and N501Y, found in variant B.1.351 (Beta). Addition of Q498H to B.1.617.1/3 increased affinity by around three-fold for ACE2, decreasing K D from 9.1nM to 2.7nM (Fig 4a). PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Q498H and Q498R mutations modify binding of B.1.351 and B.1.617.1/3 variant RBD to HuACE2. (a) Biolayer interferometry was performed with the indicated RBD in solution and HuACE2 immobilise on the sensor. Binding was measured with RBD concentrations of 12, 30, 60, 120nM, and an additional 6nM concentration in some cases. Curves were fitted and used to calculate K on , K off and K D . Data are shown as means and SEM for at least three independent experiments. (b) H498 and Y501 in SARS-CoV-2 RBD compete for interaction with Y41 in HuACE2 and the clash between residues is indicated by the overlapping Van der Waals surfaces. The proximity of the H498 side chain is shown with respect to the side chain of a Y501 inserted into our RBD:ACE2 complex structure. (c) R498 and Y501 side chain positions shown along with D38, Y41 and Q42 (PDB accession number 7BH9 [14]). https://doi.org/10.1371/journal.ppat.1010733.g004 Surprisingly, addition of Q498H to the B.1.351 variant RBD markedly reduced the affinity of B.1.351 (Fig 4a). As shown in Fig 4b, H498 and Y501 are in close proximity and the local arrangement required to accommodate the phenolic group of Y501 will be constrained by the imidazole of H498, reducing the advantageous π-π stacking between Y501 and Y41 in B.1.351 as well as perturbing the H498-Y41 interactions. Thus, the negative epistasis between Y501 and H498 results from this conflict of their side chain ring structures. As introduction of a histidine at position 498 clashes with Y501, we tested the effect of an arginine mutation at 498 (Fig 4a), as this is also a basic residue and the Q498R mutation is found in some SARS-CoV-2 variants [12]. Addition of Q498R to the N501Y-containing variant increased affinity 3.8-fold, yielding an K D of 1.6nM. This increase in affinity is due to a large decrease in off-rate, indicating the Q498R mutation in the N501Y variant acts to stabilize binding to ACE2. The increase in affinity found on addition of Q498R to the N501Y-containing variant indicates that arginine can be accommodated at position 498 without clashing with Y501, allowing both Y501 and R498 to contribute to binding. Indeed, in the absence of any other mutations RBD with the double mutation Q498R plus N501Y has very much higher affinity for ACE2 than RBD with either N501Y alone or Q498R alone (Fig 4a). To gain insight into the molecular mechanism for the affinity gain afforded by the N501Y plus Q498R mutations we examined a previously published RBD:ACE2 structure [14] in which RBD has Q498R and N501Y mutations, in addition to several other mutations. This structure suggests RBD Y501 makes a π-interaction with ACE Y41 and RBD R498 forms a hydrogen bond with ACE Q42 and a potential salt bridge with ACE D38, and could also form a cation- π interaction with Y41 (Fig 4c). Therefore, the mechanism by which the combined mutations of Q498R plus N501Y increases affinity has similarities to that by which the single Q498H mutation increases affinity. Overall, these data show that the affinity of B.1.617.1/3 variant RBD for ACE2 is enhanced by the addition of the Q498H mutation. In contrast, in a variant which already has an N501Y mutation, the Q498H mutation clashes structurally with Y501 and fails to increase affinity. However, arginine can be accommodated at 498 in a N501Y variant, and this results in a large affinity gain by a bonding mechanism requiring both R498 plus Y501, that shares similarities with that of H498 alone.

The Q498H mutation enables RBD binding to rat ACE2 RBD of SARS-CoV-2 Wuhan-Hu-1 is unable to bind rodent ACE2. However, in a recent study to develop a mouse model of SARS-CoV-2 infection, SARS-CoV-2 was passaged multiple times through mice to derive a variant capable of murine infection [15]. The resultant variant had two mutations in RBD, Q493K and Q498H, and was capable of binding to mouse ACE2 and causing SARS-CoV-2 infection [15]. Interestingly, either of these mutations alone appears sufficient to allow RBD to bind mouse ACE2. This finding suggests that SARS-CoV-2 variants that include a Q498H mutation would be able to bind mouse ACE2 and allow the virus to extend its species range of infectivity. We therefore tested whether the high affinity RBD variant S477N/Q498H-RBD can bind rodent ACE2. Given the similarity between mouse and rat ACE2 (RaACE2) in the region corresponding to the SARS-CoV-2 binding site (Fig 5b), and the importance of rats as a potential viral reservoir, we focussed on testing binding to rat ACE2. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 5. The Q498H mutation enables binding of SARS-CoV-2 RBD to rat ACE2. (a) Kinetic binding constants for RBD binding to RaACE2 measured using biolayer interferometry. Binding was measured with RBD concentrations of 48, 90, 150, 300 and 600nM. Where no binding was detected an additional 1200nM RBD concentration was also tested for binding. RBD with the Q498H mutation binds rat receptor. Data are shown as means and SEM for at least three independent experiments. (b) Rat (RaACE2) and mouse ACE2 (MoACE2) differ from human ACE2 (HuACE2) in key residues contributing to SARS-CoV-2 RBD binding. Residues in human ACE2 at the RBD binding interface are aligned with the corresponding residues in rat and mouse ACE2. Residues that form hydrogen bonds or salt bridges with RBD are in bold and those that differ in rodent are in red. (c) Bonding interactions between Q24 and N487 (d) D30 and K417 (e) Y83 and N487 plus Y489, and (f) K353 and G496, in HuACE2 (yellow) and RBD (green). https://doi.org/10.1371/journal.ppat.1010733.g005 Like the situation with mouse ACE2, we could not detect any binding between WH-RBD and RaACE2 (Fig 5a). However, we found that S477N/Q498H-RBD was clearly capable of binding RaACE2, with a K D of 28.7nM, which is comparable to the affinity of Wuhan-Hu-1 RBD for human ACE2 (Fig 2c). RBD with the S477N mutation alone was unable to bind the rodent receptor, whereas RBD with just the Q498H mutation bound with an affinity of 39.3nM (Fig 5a).

Mechanisms of binding to rodent ACE2 To understand why Wuhan-Hu-1 can bind human ACE2 but not the rodent receptor, we identified which HuACE2 residues involved in RBD binding differ in rat ACE2. Binding of RBD to HuACE2 involves three key regions of ACE2, the alpha helix between residues 19 and 42, residues 82 and 83 and residues 353 to 357 [13,16,17]. Alignment of the ACE2 sequences encompassing these regions for human and rat reveals differences between the species (Fig 5b). Four residue positions in HuACE2 identified as being involved in salt bridge or H bonding with SARS-CoV-2 RBD differ in the rat sequence, namely Q24, D30, Y83 and K353 in HuACE2 vs N24, N30, F83 and H353 in RaACE2 (Fig 5b). Of these, Q24 in HuACE2 hydrogen bonds with RBD N487 (Fig 5c), and this hydrogen bond will also likely be formed with N24 in the rat. In contrast, D30 in HuACE2 forms a salt bridge with RBD K417 (Fig 5d) and this would not occur with the corresponding N30 in rat ACE2. Y83 in HuACE2 forms two hydrogen bonds with N487 and Y489 (Fig 5e) and these would also be abolished in rat ACE2 where position 83 is a phenylalanine. K353 in HuACE2 hydrogen bonds with G496 (Fig 5f), however position 353 in RaACE2 is a histidine and the shorter reach and lower mobility of the histidine side chain would limit its to hydrogen bond with G496. Overall, the one fewer salt bridge and at least two fewer H bonds that RaACE2 could form with WH-RBD, compared with HuACE2 binding to WH-RBD, could result in the different abilities of rat and human ACE2 to bind WH-RBD. Like rat, mouse ACE2 also has N30, F83 and H353 (Fig 5b) and therefore, as with RaACE2, would form one fewer salt bridge and at least two fewer hydrogen bonds than can HuACE2 with WH-RBD, which could account for the lack of detectable binding of mouse ACE2 to WH-RBD. The ability of Q498H RBD to bind RaACE2 indicates RaACE2 can form inter-molecular bonds with H498 that are not available to Q498 in WH-RBD. With HuACE2, H498 forms bonds with D38, Y41 and K353, leading to the increase affinity of the H498 mutant over WH-RBD for the human receptor (see above). D38 and Y41 are also present in rat ACE2 (Fig 5b). Therefore, the H498 could form a π-interaction with RaACE2 Y41 and a hydrogen bond with RaACE2 D38, as in HuACE2, providing additional binding energy for the Q498H binding. In addition, in HuACE2, K353 makes an intramolecular salt bridge with D38 [13]. The shorter H353 in RaACE2 would not be able to do this, leaving D38 available for possible formation of a salt bridge with H498 in the mutant RBD. These new bonding possibilities afforded by H498 in the mutant RBD could at least partially compensate for the bonds lacking from N30 and F83 in RaACE2, bringing binding of the mutant RBD to RaACE2 up to a detectable level. D38, Y41 and H353 are also conserved in mouse ACE2 (Fig 5b), potentially allowing H498 to form similar interactions and enable the RBD to bind murine ACE2 [15].

[END]
---
[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010733

Published and (C) by PLOS One
Content appears here under this condition or license: Creative Commons - Attribution BY 4.0.

via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/