JOURNAL OF VIROLOGY, June 2011, p. 5331–5337
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 11
A Virus-Binding Hot Spot on Human Angiotensin-Converting Enzyme 2
Is Critical for Binding of Two Different Coronaviruses?
Kailang Wu,1Lang Chen,1Guiqing Peng,1Wenbo Zhou,2Christopher A. Pennell,3
Louis M. Mansky,4Robert J. Geraghty,2and Fang Li1*
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 554551; Center for Drug Design,
University of Minnesota, Minneapolis, Minnesota 554552; Cancer Center, Center for Immunology, University of Minnesota,
Minneapolis, Minnesota 554553; and Institute for Molecular Virology and Departments of Diagnostic and
Biological Sciences and Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 554554
Received 29 October 2010/Accepted 2 March 2011
How viruses evolve to select their receptor proteins for host cell entry is puzzling. We recently determined
the crystal structures of NL63 coronavirus (NL63-CoV) and SARS coronavirus (SARS-CoV) receptor-binding
domains (RBDs), each complexed with their common receptor, human angiotensin-converting enzyme 2
(hACE2), and proposed the existence of a virus-binding hot spot on hACE2. Here we investigated the function
of this hypothetical hot spot using structure-guided biochemical and functional assays. The hot spot consists
of a salt bridge surrounded by hydrophobic tunnel walls. Mutations that disturb the hot spot structure have
significant effects on virus/receptor interactions, revealing critical energy contributions from the hot spot
structure. The tunnel structure at the NL63-CoV/hACE2 interface is more compact than that at the SARS-
CoV/hACE2 interface, and hence RBD/hACE2 binding affinities are decreased either by NL63-CoV mutations
decreasing the tunnel space or by SARS-CoV mutations increasing the tunnel space. Furthermore, NL63-CoV
RBD inhibits hACE2-dependent transduction by SARS-CoV spike protein, a successful application of the hot
spot theory that has the potential to become a new antiviral strategy against SARS-CoV infections. These
results suggest that the structural features of the hot spot on hACE2 were among the driving forces for the
convergent evolution of NL63-CoV and SARS-CoV.
Host receptor recognition by viruses is the first and essential
step for viral infections. During the long history of evolutionary
battles between viruses and hosts, viruses have evolved com-
plex strategies for their receptor selections (2). Despite tre-
mendous efforts to understand these strategies, the current
picture of how viruses recognize their host receptors is still
murky. Viruses exploit a wide variety of host cell surface mol-
ecules as their receptors. In addition to serving as receptors for
viruses, these molecules are implicated in various host physi-
ological functions such as cell adhesion, immune response,
signaling pathways, proteolysis, and ion transport. On one
hand, several viruses can share one host receptor. For example,
coxsackievirus-adenovirus receptor, an immunoglobulin (Ig)
superfamily member, is the receptor for both coxsackieviruses
and adenoviruses (3). On the other hand, one virus can recog-
nize several different host receptors. For example, herpes sim-
plex viruses use one of at least three protein receptors: HVEM,
which is a tumor necrosis factor receptor family member (23),
and nectin-1 and nectin-2, both of which are Ig superfamily
members (8, 31). Understanding the pattern of host receptor
recognition by viruses has important implications for viral evo-
lution, pathogenesis, host range, tropism, cross-species infec-
tions, emerging viral epidemics, and virus-mediated gene tar-
A key question regarding the evolution of host receptor
recognition by viruses is what features of these receptor mol-
ecules make them targeted by viruses. The receptors for vi-
ruses can be proteins, carbohydrates, or lipids (2). Compared
with carbohydrates and lipids, protein receptors in general
have more structural features and thus are engaged in more-
specific and high-affinity binding interactions with viruses; they
are the focus of this study. Among protein receptors, some
(such as cell adhesion molecules) are more abundant than
others (such as proteases and ion transporters). Although the
availability of abundant protein receptors to viruses is probably
one of the reasons why they were selected by viruses as recep-
tors (30), it is not clear whether receptor proteins, especially
nonabundant receptor proteins, contain any structural features
that make them targeted by viruses.
The structural features of receptor proteins can be identified
from the atomic structures of virus/receptor interfaces. De-
fined structural and functional receptor-binding domains
(RBDs) have been identified in many viral surface glycopro-
teins. One or more receptor-binding motifs (RBMs) on these
viral RBDs mediate the interactions with their receptor pro-
teins. To date several crystal structures are available for viral
RBDs complexed with their receptor proteins (1, 4, 5, 13, 18,
32, 33). Among these structures, only two reveal how different
viral RBDs can bind to their common receptor protein: the
structures of NL63 coronavirus (NL63-CoV) and SARS coro-
navirus (SARS-CoV) RBDs, each complexed with their
common receptor, human angiotensin-converting enzyme 2
(hACE2) (18, 32). Both NL63-CoV and SARS-CoV are im-
portant human viral pathogens. The former causes prevalent
respiratory diseases (6, 29), whereas the latter was responsible
* Corresponding author. Mailing address: Department of Pharma-
cology, University of Minnesota Medical School, 6-121 Jackson Hall,
321 Church St. SE, Minneapolis, MN 55455. Phone: (612) 625-6149.
Fax: (612) 625-8408. E-mail: email@example.com.
?Published ahead of print on 16 March 2011.
for the worldwide epidemic of severe acute respiratory syn-
drome (SARS) diseases in 2002 to 2003 (12, 24). Coronavirus
spike glycoproteins are envelope-anchored clove-shaped tri-
mers (16). Each spike trimer contains three monomeric S1
heads, which function in receptor binding, and a trimeric S2
stalk, which functions in fusing the viral envelope and host
membrane. NL63-CoV and SARS-CoV RBDs are located in
the S1 heads of their respective spike proteins. There is no
structural homology in their RBD core structures or RBMs
(Fig. 1). The core structures of NL63-CoV and SARS-CoV
RBDs are a two-layer ?-sandwich and a single-layer ?-sheet,
respectively; the RBMs of NL63-CoV and SARS-CoV are
three discontinuous short loops and one continuous long
subdomain, respectively. Nevertheless, the two viral RBDs
bind to the same three virus-binding motifs (VBMs) on
hACE2 (18, 32).
Our previous structural studies led to the hypothesis that a
virus-binding hot spot exists on hACE2 and plays an important
role in the binding of both NL63-CoV and SARS-CoV (32).
This hypothetical hot spot consists of a critical Lys353-Asp38
salt bridge on hACE2, which is surrounded by four hydropho-
bic tunnel walls (Fig. 2A and B). Two of the tunnel walls, Tyr41
(top wall) and Asp37 (right wall), are contributed by hACE2,
whereas the other two tunnel walls are contributed by the
viruses: Tyr498 (bottom wall) and Ser535 (left wall) from
NL63-CoV and Tyr491 (bottom wall) and Thr487 (left wall)
from SARS-CoV. Details of how this hypothetical hot spot
may contribute to the virus/receptor interactions are unknown.
In this study we use structure-guided biochemical and func-
tional approaches to investigate the role of each of the com-
ponents of the hot spot structure in the virus/receptor interac-
tions. We then apply the hot spot theory to the development of
FIG. 1. Overall structures of NL63-CoV and SARS-CoV RBDs, each complexed with their common receptor, human ACE2. (A) Crystal
structure of NL63-CoV RBD complexed with hACE2 (PDB 3KBH). hACE2 is green, virus-binding motifs (VBMs) are blue, the NL63-CoV RBD
core structure is cyan, and receptor-binding motifs (RBMs) are red. Lys353 and Asp38 in hACE2, which are critical for the RBD/hACE2
interactions, are shown in ball-and-stick format. (B) Crystal structure of SARS-CoV RBD complexed with hACE2 (PDB 2AJF).
FIG. 2. Detailed structure of a common virus-binding hot spot on human ACE2. (A) Hot spot structure at the NL63-CoV/hACE2 interface.
(B) Hot spot structure at the SARS-CoV/hACE2 interface. (C) Conformation of Lys353 on the surface of unbound human ACE2 (PDB 1R42).
5332 WU ET AL. J. VIROL.
a potentially new antiviral strategy against SARS-CoV infec-
tions. We also discuss how the structural features of the hot
spot drove the convergent evolution of two different viruses.
MATERIALS AND METHODS
Protein expression and purification. Soluble proteins, including NL63-CoV
RBD (residues 461 to 616), SARS-CoV RBD (residues 306 to 527), and hACE2
peptidase domain (residues 19 to 615), were expressed and purified as previously
described (17, 18, 32). In brief, the proteins were expressed in insect cells using
the Bac-to-Bac system (Life Technologies Inc.). Each expression construct (In-
vitrogen) contained an N-terminal honeybee melittin signal sequence and a
C-terminal His tag sequence. Mutations were introduced by PCR site-directed
mutagenesis to the expression constructs. Recombinant baculoviruses were gen-
erated and amplified in Sf9 insect cells. The protein to be purified was harvested
from Sf9 cell supernatants, loaded onto a Ni-nitrilotriacetic acid (Ni-NTA)
column, eluted from the Ni-NTA column with 0.5 M imidazole, and further
purified by gel filtration chromatography on Superdex 200 (GE Healthcare).
Fractions containing the purified protein were pooled together, loaded into an
Amicon ultra-15 centrifugal filter unit (10,000-molecular-weight [MW] cutoff)
(Millipore), and centrifuged at 10,000 rpm until the protein concentration
reached 10 mg/ml.
RBD/hACE2 binding assays. Surface plasmon resonance assays were carried
out using a Biacore 2000 as previously described (32). In brief, to measure the
affinities for binding between mutant viral RBDs and wild-type hACE2, hACE2
was immobilized on a C5 sensor chip through direct covalent coupling via amine
groups. The surface of the sensor chip was activated with N-hydroxysuccinimide
(NHS), the receptor was injected and immobilized to the surface of the chip, and
the remaining activated surface of the chip was blocked with ethanolamine.
Soluble RBDs were introduced at a flow rate of 20 ?l/min at different concen-
trations (62.5 nM, 125 nM, and 250 nM). The on rate (kon), the off rate (koff), and
the dissociation constant (Kd) were determined for the RBD/receptor binding
interactions using BIA-EVALUATIONS software. To measure the affinities for
binding between mutant hACE2 and wild-type viral RBDs, RBDs were immo-
bilized on the sensor chip and hACE2 was the soluble analyte. As negative
controls, soluble RBDs or hACE2 was passed through an empty sensor chip and
buffer alone was passed through sensor chips containing RBDs or hACE2 as
Transduction assays with pseudotyped virus. Transduction was assayed using
murine leukemia viruses (MLVs) expressing ?-galactosidase and pseudotyped
with NL63-CoV or SARS-CoV spike protein. To prepare pseudotyped viruses,
HEK293T cells were cotransfected with spike protein-encoding pcDNA3.1 and
MLV ?-galactosidase-transducing vector pBAG (25). At 2 days posttransfection,
viral supernatants were harvested and concentrated in a spin concentrator. Ap-
proximately 4 ml of supernatant was typically concentrated (10,000-MW cutoff)
to between 100 to 200 ?l. HEK293T cells transiently expressing hACE2 in
pcDNA3.1 were inoculated in 96-well dishes by adding 5 ?l of concentrated viral
supernatant to 50 ?l cell culture medium per well. Transduction efficiency was
quantified 2 days later by measuring ?-galactosidase activity. The inoculated cells
were lysed in phosphate-buffered saline (PBS) containing 0.5% NP-40 and 3
mg/ml o-nitrophenyl-?-D-glucopyranoside and monitored by spectrometry (op-
tical density at 410 nm [OD410]). The intracellular C termini of the spike protein
and hACE2 contained a C9 tag and a hemagglutinin (HA) tag, respectively. The
concentrations of the spike protein packaged in pseudotyped viruses and of
hACE2 expressed on the HEK293T cell surface were detected by Western
blotting using anti-C9 and anti-HA antibodies, respectively. As a negative con-
trol, the plasmid expressing the spike protein was replaced by a plasmid that does
not express any protein.
To investigate the role of the hot spot structure in the virus/
receptor binding interactions, we mutated each of the compo-
nents 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 bind-
ing between the wild-type hACE2 peptidase domain and pro-
totypic 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 Bia-
core 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 Kdfor SARS-
CoV RBD and hACE2 binding was 20.8 nM (Fig. 3A), con-
sistent with the Kdof 16.2 nM measured in a previous study
(19). The measured Kdfor NL63-CoV RBD and hACE2 bind-
ing was 34.9 nM (Fig. 3A), the first reported Kdfor 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 Kds for binding with hACE2, NL63-
CoV RBD bound to hACE2 with significantly lower koffand
kon. It has been shown that koffand konare dictated by short-
range van der Waals interactions and long-range electrostatic
interactions between the proteins, respectively (26). Therefore,
the lower koffand konof the NL63-CoV-RBD/hACE2 complex
likely reflected a less electrostatic and more hydrophobic in-
terface between the two proteins.
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
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 binding
interaction between wild-type hACE2 and wild-type RBDs. (B) Bia-
core analyses of NL63-CoV RBD and hACE2. Single mutations were
introduced to hACE2 or RBD to modify every component of the hot
spot structure. Ka, association constant. (C) Biacore analyses of
SARS-CoV RBD and hACE2.
VOL. 85, 2011NL63 AND SARS VIRUSES TARGET A HOT SPOT ON HUMAN ACE25333
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 (Fig. 3A). Such
discrepancies in measured Kdwere mostly due to the differ-
ences in measured kon. No matter whether hACE2 or RBDs
were immobilized, koffs remained similar. When hACE2 was
immobilized, however, konwas significantly higher. Why did
konincrease 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 intro-
duced single mutations to either RBDs or hACE2 that modi-
fied every component of the hot spot structure. These muta-
tions 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 (Fig. 2A and B). We
expressed and purified each of the 11 hACE2 and RBD mu-
tants. 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 immobi-
lized 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 (Fig.
3B and C).
In this study we not only measured direct interactions be-
tween 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 ?-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 mea-
sured by determining ?-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 muta-
tions used for Biacore assays (Fig. 4A and B). The expression
levels of the spike proteins in pseudotyped viruses and of
hACE2 molecules on HEK293T cells were detected by West-
ern blotting using antibodies against their intracellular C-ter-
minal 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 (Fig. 4C). 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).
Both Biacore assays and transduction assays with pseu-
dotyped virus yielded results that were highly consistent with
each other (Fig. 3 and 4; Tables 1 and 2). 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
FIG. 4. Transduction assays with pseudotyped virus of the interac-
tions between viral spike proteins and human ACE2. Retroviral MLVs
expressing ?-galactosidase and pseudotyped with the NL63-CoV or
SARS-CoV spike protein were used to infect hACE2-expressing
HEK293T cells. Transduction efficiency of the pseudotyped viruses
was measured by ?-galactosidase assays. After mutations were intro-
duced into the spike proteins or hACE2, the corresponding transduc-
tion efficiency was normalized against the transduction efficiency of
viruses pseudotyped with wild-type spike proteins in cells expressing
wild-type hACE2. Each experiment was repeated 6 times. The corre-
sponding standard errors are shown. (A) Transduction of MLVs pseu-
dotyped with NL63-CoV spike protein in hACE2-expressing cells.
(B) Transduction of MLVs pseudotyped with SARS-CoV spike pro-
tein in hACE2-expressing cells. (C) Western blotting of coronavirus
spike proteins and hACE2. The NL63-CoV and SARS-CoV spike
proteins packaged in pseudotyped retroviruses both contained a C-ter-
minal C9 tag, and the hACE2 expressed on the HEK293T cell surface
contained a C-terminal HA tag. The expression levels of the spike
proteins and hACE2 were detected by Western blotting using anti-C9
and anti-HA antibodies, respectively. The protein bands were quanti-
fied using software Image J (version 1.6).
5334WU ET AL. J. VIROL.
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 tar-
geted mutations produced significantly reduced RBD/hACE2
binding affinities and spike-guided transduction compared with
those for the corresponding wild-type proteins (t test; P ? 0.01
for both Kaand transduction), with the exception of D37A in
hACE2 (P ? 0.075 for transduction) and Y498 in NL63-CoV
RBD (P ? 0.10 for both Kaand transduction). Here we com-
bine 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.
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 hydro-
phobic environment, the salt bridge not only provides a signif-
icant amount of energy to the virus/receptor binding interac-
tions 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 (Fig. 3 and 4). The hydrophobic tunnel
walls of the hot spot structure also make important contribu-
tions 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 (Fig. 3 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 aro-
matic ring. Alanine substitution for Tyr41 significantly de-
creased 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 af-
finities and viral transductions. Detailed structural analysis re-
veals 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 (Fig. 2A 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 an-
chored 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 inter-
The hot spot structures at the NL63-CoV/hACE2 and
SARS-CoV/hACE2 interfaces differ in a subtle but function-
ally important way. The tunnel structure at the NL63-CoV/
hACE2 interface is more compact than that at the SARS-CoV/
hACE2 interface (Fig. 2A and B). At the NL63-CoV/hACE2
interface, the closest distances between the two pairs of op-
posing 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 threo-
nine at the 487 position in SARS-CoV RBD, these distances
become 9.0 Å and 8.1 Å, respectively. Because of the compact-
ness of the tunnel structure at the NL63-CoV/hACE2 inter-
face, S535T mutation in NL63-CoV RBD decreased the tunnel
space and was energetically unstable (Fig. 3B and 4A). 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 energeti-
cally unstable (Fig. 3C and 4B). 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, 19, 27). 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 struc-
ture as the bottom tunnel wall in a more spacious tunnel space,
TABLE 1. Summary of Biacore and pseudotyped-virus transduction
data from Fig. 3 and 4, with RBDs immobilized
Dataafor interaction with RBD of:
aValues are means (?standard errors). Ka, association constant.
bWT, wild type.
cToo low, too low for measurement.
TABLE 2. Summary of Biacore and pseudotyped-virus transduction
data from Fig. 3 and 4, with hACE2 immobilized
Virus and form
Dataafor interaction with hACE2
aValues are means (?standard errors). Ka, association constant.
bWT, wild type.
cToo low, too low for measurement.
VOL. 85, 2011NL63 AND SARS VIRUSES TARGET A HOT SPOT ON HUMAN ACE25335
and hence alanine substitution for Tyr491 decreased RBD/
hACE2 binding affinities and viral transductions (Fig. 3 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 (Fig. 5). Transduction was shown as
a percentage of ?-galactosidase activity observed in the ab-
sence of any inhibitor. The results showed that NL63-CoV
RBD indeed inhibited SARS-CoV spike-mediated transduc-
tions. At 10 ?g/ml (0.47 ?M), 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
Binding to the same hot spot on hACE2 was likely an out-
come of convergent evolution by NL63-CoV and SARS-CoV.
Our study provides several lines of evidence to support this
notion. First, despite no structural homology in their RBDs,
NL63-CoV and SARS-CoV both bind to the hot spot region
and form highly similar and energetically stable tunnel struc-
tures (Fig. 1 and 2). Second, despite no structural homology in
their RBMs, both viruses insert an RBM loop between VBM2
and VBM3 on hACE2 (Fig. 1). Third, despite being presented
by nonhomologous RBM loops, Ser535 in NL63-CoV RBD
and Thr487 in SARS-CoV RBD (Ser487 in RBDs of some
low-pathogenicity SARS-CoV strains) occupy identical posi-
tions as the left tunnel wall in the hot spot structure (Fig. 2A
and B) and contribute energy to the virus/receptor binding
interactions (Fig. 3 and 4). Last, despite pointing in opposite
directions, Tyr498 in NL63-CoV RBD and Tyr491 in SARS-
CoV RBD also occupy identical positions as the bottom tunnel
wall in the hot spot structure (Fig. 2A and B). These data
suggest that NL63-CoV and SARS-CoV likely evolved inde-
pendent strategies to achieve the same functional goal, sup-
porting a convergent evolutionary relationship between the
The likely convergent evolution of NL63-CoV and SARS-
CoV was at least partly driven by the structural features of the
virus-binding hot spot on hACE2. The general structural fea-
tures of the hot spot favor virus binding: it is located in a region
on hACE2 that is furthest from the membrane, relatively flat,
free of glycosylation, and thereby easily accessible to viruses
(Fig. 1). The detailed structural features of the hot spot, such
as its potential to form the energetically stable tunnel struc-
ture, also favor virus binding (Fig. 2A and B). In the unbound
hACE2 structure, where structural restraints from viruses are
absent, Lys353 projects into solution; it does not form a salt
bridge with Asp38 or stack with Tyr41 or Asp37 (Fig. 2C) (15,
28). Thus, the virus-binding hot spot is not preexistent or
preorganized on hACE2; instead, it is induced to form by virus
binding. Therefore, while the hot spot is mainly an intrinsic
property of hACE2, it is also a dynamic structure and receives
structural contributions from both hACE2 and the viruses,
although the contributions from hACE2 are more pronounced.
The virus-binding hot spot on hACE2 is likely different from
the hot spots for host protein/protein interactions. Host pro-
tein partners have coevolutionary relationships (9, 11), and
hence hot spots for host protein/protein interactions are usu-
ally preexistent and preorganized in unbound host protein
structures (20, 22). Viruses and receptors, however, do not
usually have such coevolutionary relationships; viruses evolve
to adapt to host receptors, but receptors do not evolve to adapt
to viruses. Occasionally, however, if a virus exerts a large
enough impact on a host, the host receptor may evolve away
from virus binding (7). So far the virus-binding hot spot on
hACE2 is not known for its interaction with any other host
proteins. Overall, the potential of the hot spot region on
hACE2 to form energetically stable tunnel structures and some
general structural features of this region on the receptor sur-
face were among the possible reasons why the hot spot was
exploited by two different viruses.
We thank Lorraine Albritton for the pBAG vector and Michael
Farzan for spike protein genes.
This work was supported by NIH grant R01AI089728 (to F.L.) and
by a University of Minnesota AHC Faculty Research Development
Grant (to F.L. and L.M.M.).
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 RBD containing the T487S mutation, hACE2 (pos-
itive control), and bovine serum albumin (BSA; negative control).
Transduction is shown as a percentage of ?-galactosidase activity ob-
served in the absence of any inhibitor. Each experiment was repeated
5 times. The corresponding standard errors are shown. The results
show that NL63-CoV can efficiently inhibit SARS-CoV spike-medi-
5336 WU ET AL.J. VIROL.
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