To investigate the role of the hot spot structure in the virus/receptor binding interactions, we mutated each of the components of the hot spot structure. We then examined how the mutations affect the affinities for binding between RBDs and hACE2 using surface plasmon resonance Biacore assays. We also investigated how the mutations impact the interactions between spike proteins and hACE2 by transduction assays using pseudotyped virus.
For Biacore assays, we first measured the affinities for binding between the wild-type hACE2 peptidase domain and prototypic NL63-CoV RBD (strain Amsterdam 1) and between the wild-type hACE2 peptidase domain and prototypic SARS-CoV RBD (strain Tor2, which was isolated during the 2002 to 2003 SARS epidemic). hACE2 was immobilized on the Biacore sensor chip through direct covalent coupling via amine groups, and NL63-CoV or SARS-CoV RBD was injected over the chip as the soluble analyte. The measured Kd
for SARS-CoV RBD and hACE2 binding was 20.8 nM (), consistent with the Kd
of 16.2 nM measured in a previous study (19
). The measured Kd
for NL63-CoV RBD and hACE2 binding was 34.9 nM (), the first reported Kd
for binding between the two proteins. The same RBD fragment used in this study (residues 461 to 616) also bound to hACE2 with high affinity in a previous study using a coimmunoprecipitation analysis (21
). Interestingly, although SARS-CoV and NL63-CoV RBDs had similar Kd
s for binding with hACE2, NL63-CoV RBD bound to hACE2 with significantly lower koff
. It has been shown that koff
are dictated by short-range van der Waals interactions and long-range electrostatic interactions between the proteins, respectively (26
). Therefore, the lower koff
of the NL63-CoV-RBD/hACE2 complex likely reflected a less electrostatic and more hydrophobic interface between the two proteins.
Fig. 3. Surface plasmon resonance Biacore analyses of the binding interactions between viral RBDs and human ACE2. Each experiment was repeated 5 times at three different protein concentrations. The corresponding standard errors are shown. (A) Kinetics of the (more ...)
Using Biacore, we also measured the affinities for binding between hACE2 and NL63-CoV RBD and between hACE2 and SARS-CoV RBD in a reverse way: NL63-CoV or SARS-CoV RBD was immobilized on the sensor chip, and hACE2 was injected over the chip as the soluble analyte. The measured Kds were 68.0 nM for NL63-CoV RBD and hACE2 and 137 nM for SARS-CoV RBD and hACE2, both of which were higher than when hACE2 was immobilized (). Such discrepancies in measured Kd were mostly due to the differences in measured kon. No matter whether hACE2 or RBDs were immobilized, koffs remained similar. When hACE2 was immobilized, however, kon was significantly higher. Why did kon increase when hACE2, instead of RBDs, was immobilized? This is because hACE2 has a larger molecular weight than either of the RBDs, and thus when immobilized, hACE2 can provide more accessible surface area for complex formation, leading to higher kon. Therefore, the surface accessibility of the immobilized protein, but not the dissociation rate, accounted for the discrepancies in measured Kd.
To evaluate how mutations of the hot spot structure affect the affinities for binding between RBDs and hACE2, we introduced single mutations to either RBDs or hACE2 that modified every component of the hot spot structure. These mutations were K353A, D38A, D37A, Y41A, and Y41F in hACE2, Y498A, S535A, and S535T in NL63-CoV RBD, and Y491A, T487A, and T487S in SARS-CoV RBD ( and B). We expressed and purified each of the 11 hACE2 and RBD mutants. To measure the affinities for binding between mutant RBDs and wild-type hACE2, hACE2 was immobilized on the sensor chip and mutant RBDs were the soluble analytes. To measure the affinities for binding between wild-type RBDs and mutant hACE2, NL63-CoV or SARS-CoV RBD was immobilized on the sensor chip and mutant hACE2 was the soluble analyte. The results were then compared with the affinities for binding between wild-type hACE2 and wild-type RBDs ( and C).
In this study we not only measured direct interactions between viral RBDs and hACE2 using recombinant proteins but also examined the spike/receptor interactions using functional assays. To this end, we carried out transduction assays with pseudotyped virus to investigate whether changes in RBD/hACE2 interactions could lead to corresponding changes in viral entry and membrane fusion in the context of the full-length spike proteins and their receptor protein. We prepared retroviral MLVs expressing β-galactosidase and pseudotyped with NL63-CoV or SARS-CoV spike protein. These MLVs were incubated with hACE2-expressing HEK293T cells. The transduction efficiency of the pseudotyped viruses was measured by determining β-galactosidase activity of inoculated cell lysate. To measure the interactions between wild-type hACE2 and mutant spike proteins or between mutant hACE2 and wild-type spike proteins, we introduced single mutations to hACE2 or the spike proteins that were the same as the mutations used for Biacore assays ( and B). The expression levels of the spike proteins in pseudotyped viruses and of hACE2 molecules on HEK293T cells were detected by Western blotting using antibodies against their intracellular C-terminal C9 and HA tags, respectively. The Western blotting results showed that all of the mutant spike proteins and mutant hACE2 molecules were well expressed, and the expression levels of these mutant proteins were quantified and calibrated against those of the wild-type proteins (). Finally, the measured transduction efficiencies for mutant spike proteins and mutant hACE2 were normalized against the transduction efficiency of viruses pseudotyped with wild-type spike proteins in cells expressing wild-type hACE2 ().
Fig. 4. Transduction assays with pseudotyped virus of the interactions between viral spike proteins and human ACE2. Retroviral MLVs expressing β-galactosidase and pseudotyped with the NL63-CoV or SARS-CoV spike protein were used to infect hACE2-expressing (more ...)
Both Biacore assays and transduction assays with pseudotyped virus yielded results that were highly consistent with each other ( and ; and ). It is worth noting that recombinant SARS-CoV and NL63-CoV RBDs are both monomers in solution, whereas the full-length spike proteins are trimers on virus surfaces (14
). Thus, the good correlation between the RBD/hACE2 binding affinities and the spike-mediated transduction efficiency strongly suggests that the measured RBD/hACE2 binding activities reflect the native states of the proteins. Our results showed that all of the targeted mutations produced significantly reduced RBD/hACE2 binding affinities and spike-guided transduction compared with those for the corresponding wild-type proteins (t
< 0.01 for both Ka
and transduction), with the exception of D37A in hACE2 (P
> 0.075 for transduction) and Y498 in NL63-CoV RBD (P
> 0.10 for both Ka
and transduction). Here we combine these biochemical and functional data with our previous structural data and discuss molecular and structural features of the virus-binding hot spot that make hACE2 a common target by two different viruses.
Summary of Biacore and pseudotyped-virus transduction data from and , with RBDs immobilized
Summary of Biacore and pseudotyped-virus transduction data from and , with hACE2 immobilized
The hot spot structures at the NL63-CoV/hACE2 and SARS-CoV/hACE2 interfaces have many common features. The Lys353-Asp38 salt bridge plays a central role in the hot spot structure at both of the interfaces. Because of the hydrophobic environment, the salt bridge not only provides a significant amount of energy to the virus/receptor binding interactions but also fills a critical void in the hydrophobic stacking interactions at the virus/receptor interfaces. Correspondingly, alanine substitution for either Lys353 or Asp38 in hACE2 significantly decreased the RBD/hACE2 binding affinities and viral transductions ( and 4). The hydrophobic tunnel walls of the hot spot structure also make important contributions to the virus/receptor binding interactions; they not only support the side chain of Lys353 to form the salt bridge but also provide hydrophobic stacking interactions at the virus/receptor interfaces. Some hydrophobic tunnel walls contribute more energy to the virus/receptor binding interactions than others ( and 4). For example, Tyr41 in hACE2 (top wall) is more important than Asp37 in hACE2 (right wall), probably because Tyr41 functions better as a tunnel wall with its aromatic ring. Alanine substitution for Tyr41 significantly decreased RBD/hACE2 binding affinities and viral transductions, suggesting that the stacking interaction between Tyr41 and Lys353 is essential for the hot spot structure. Interestingly, although a phenylalanine at the 41 position can potentially function as a tunnel wall with its aromatic ring, the Y41F mutation also significantly decreased RBD/hACE2 binding affinities and viral transductions. Detailed structural analysis reveals that the hydroxyl group of Tyr41 forms a hydrogen bond with receptor Asp355 at the NL63-CoV/hACE2 interface and two hydrogen bonds with receptor Asp355 and RBD Thr486 at the SARS-CoV/hACE2 interface ( and B). Thus, the side chain of Tyr41 needs to be firmly anchored in order for it to function properly as a tunnel wall. Residue 41 is a histidine in the ACE2 proteins from several bat species (10
). Not only is His41 a poor hydrophobic stacker, but also it cannot be anchored properly to function as a tunnel wall. As a result, these bat ACE2 proteins were poor receptors for human SARS-CoV strains unless an H41Y mutation was introduced (10
). Overall, the salt bridge and many of the tunnel walls of the hot spot structure contribute energy to the virus/receptor binding interactions.
The hot spot structures at the NL63-CoV/hACE2 and SARS-CoV/hACE2 interfaces differ in a subtle but functionally important way. The tunnel structure at the NL63-CoV/hACE2 interface is more compact than that at the SARS-CoV/hACE2 interface ( and B). At the NL63-CoV/hACE2 interface, the closest distances between the two pairs of opposing tunnel walls, Ser535-Asp37 (left to right) and Tyr41-Tyr498 (top to bottom), are 8.5 Å and 7.7 Å, respectively. At the SARS-CoV/hACE2 interface, if a serine replaces threonine at the 487 position in SARS-CoV RBD, these distances become 9.0 Å and 8.1 Å, respectively. Because of the compactness of the tunnel structure at the NL63-CoV/hACE2 interface, S535T mutation in NL63-CoV RBD decreased the tunnel space and was energetically unstable ( and ). In contrast, because of the extra space of the tunnel structure at the SARS-CoV/hACE2 interface, T487S mutation in SARS-CoV RBD increased the tunnel space but was also energetically unstable ( and ). Indeed, residue 487 was a serine in RBDs of some low-pathogenicity SARS-CoV strains and was largely responsible for the lack of human-to-human transmission of these viral strains (18
). Thus, although S535T mutation in NL63-CoV RBD and T487S mutation in SARS-CoV RBD exerted opposite effects on the same left tunnel wall of the hot spot structure, they both reduced RBD/hACE2 binding affinities and viral transductions. For similar reasons, compared with Tyr498 in NL63-CoV RBD, Tyr491 in SARS-CoV RBD provides more support to the hot spot structure as the bottom tunnel wall in a more spacious tunnel space, and hence alanine substitution for Tyr491 decreased RBD/hACE2 binding affinities and viral transductions ( and 4). Therefore, the seemingly small differences in the hot spot structure at the two virus/receptor interfaces not only have significant impacts on virus/receptor binding interactions but also have important epidemic implications.
One of the direct implications of our study is the possibility of using NL63-CoV RBD as an inhibitor to block SARS-CoV infections, because NL63-CoV RBD can compete with SARS-CoV for the common virus-binding hot spot on hACE2. To test this possibility, we inoculated MLVs pseudotyped with SARS-CoV spike protein onto hACE2-expressing HEK293T cells in the presence of various concentrations of purified NL63-CoV RBD or SARS-CoV RBD (). Transduction was shown as a percentage of β-galactosidase activity observed in the absence of any inhibitor. The results showed that NL63-CoV RBD indeed inhibited SARS-CoV spike-mediated transductions. At 10 μg/ml (0.47 μM), NL63-CoV RBD inhibited SARS-CoV spike-mediated transductions by over 80%. This method has the potential to become a new antiviral strategy against SARS-CoV infections, as it represents the first case in which SARS-CoV infection can be inhibited by a protein from a different virus. It also represents a successful application of the common virus-binding hot spot theory derived from the present study.
Fig. 5. Inhibition of SARS-CoV spike-mediated transduction by NL63-CoV RBD. MLVs pseudotyped with SARS-CoV spike protein were used to infect hACE2-expressing HEK293T cells in the presence of various concentrations of purified NL63-CoV RBD, SARS-CoV RBD, SARS-CoV (more ...)