Structural Basis for Variation in Adenovirus Affinity for the Cellular Coxsackievirus and Adenovirus Receptor*
Jason Howitt
,
Maria C. Bewley,
Vito Graziano,
John M. Flanagan and
Paul Freimuth
From the
Biology Department, Brookhaven National Laboratory, Upton, New York
11973
Received for publication, February 11, 2003
, and in revised form, April 15, 2003.
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ABSTRACT
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The majority of adenovirus serotypes can bind to the coxsackievirus and
adenovirus receptor (CAR) on human cells despite only limited conservation of
the amino acid residues that comprise the receptor-binding sites of these
viruses. Using a fluorescence anisotropy-based assay, we determined that the
recombinant knob domain of the fiber protein from adenovirus serotype (Ad) 2
binds the soluble, N-terminal domain (domain 1 (D1)) of CAR with 8-fold
greater affinity than does the recombinant knob domain from Ad12. Homology
modeling predicted that the increased affinity of Ad2 knob for CAR D1 could
result from additional contacts within the binding interface contributed by
two residues, Ser408 and Tyr477, which are not conserved
in the Ad12 knob. Consistent with this structural model, substitution of
serine and tyrosine for the corresponding residues in the Ad12 knob (P417S and
S489Y) increased the binding affinity by 4- and 8-fold, respectively, whereas
the double mutation increased binding affinity 10-fold. X-ray structure
analysis of Ad12 knob mutants P417S and S489Y indicated that both substituted
residues potentially could form additional hydrogen bonds across the knob-CAR
interface. Structural changes resulting from these mutations were highly
localized, implying that the high tolerance for surface variation conferred by
the stable knob scaffold can minimize the impact of antigenic drift on binding
specificity and affinity during evolution of virus serotypes. Our results
suggest that the interaction of knob domains from different adenovirus
serotypes with CAR D1 can be accurately modeled using the Ad12 knob-CAR D1
crystal structure as a template.
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INTRODUCTION
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Interaction of animal viruses with their cellular receptors is of
particular interest because the viral proteins that function in molecular
recognition are subject to immunoselective pressure to vary their antigenic
structure. The potentially negative impact of antigenic variation on
recognition of the cellular receptor is minimized in some virus families by
protein structural features that shield receptor-binding sites from attack by
host-neutralizing antibodies
(1). In adenoviruses, by
contrast, receptor-binding sites are exposed and highly variable in sequence.
Therefore, adenovirus-receptor interaction presents an opportunity to observe
the range of structural plasticity that can be tolerated without loss of
binding specificity.
Receptor-binding function in adenoviruses is associated with viral fibers
(2,
3), rod-shaped homotrimeric
proteins (4) that protrude from
each vertex of the icosahedral virus capsid. The distal end of viral fibers
consists of a globular domain, the head or knob domain, which has receptor
binding activity (5,
6). Human adenoviral fiber
proteins have evolved to recognize several different host cell receptors
(79);
however, to date only one of these receptors has been molecularly
characterized, a 46-kDa membrane glycoprotein known as the coxsackievirus and
adenovirus receptor
(CAR)1
(10,
11). Representative serotypes
from adenovirus subgroups A, C, D, E, and F have been shown to interact with
CAR (12), suggesting that CAR
may be the most common receptor used by human adenoviruses. The CAR protein
spans the cell plasma membrane once and has two extracellular Ig domains
derived from the N-terminal region of the polypeptide. The CAR N-terminal Ig
variable-type domain (D1) alone is necessary and sufficient for interaction
with the fiber protein knob domain
(1315).
CAR has a broad tissue distribution
(16) and is found
predominantly on the basolateral surfaces of epithelial cells
(17,
18). CAR is localized
specifically within tight junctions
(19,
20), which hold lateral cell
membranes in close apposition to form an effective seal close to the apical
surface. Although interaction of viruses with receptors such as CAR that have
low expression levels on cell apical surfaces appears paradoxical, recent
studies suggest that this arrangement could facilitate the spread of virus
infection within tissues and transmission of virus particles to new hosts,
because progeny virus particles are released preferentially from the
basal-lateral surfaces of infected cells
(19,
21).
X-ray structures of recombinant knob domains from the fiber proteins of
adenovirus serotypes 2, 3, 5, and 12 have been determined
(2225)
and in all cases show that knob monomers fold into similar 8-stranded
-sandwich conformations. Knob monomers assemble into remarkably stable
trimers (26), resulting from a
large number of hydrogen bonds and hydrophobic interactions in the noncovalent
trimer interface (23).
Variations in polypeptide chain length are accommodated by changes in the
length of surface loops, whereas other sequence variations appear to have had
minimal effects on the underlying
-sandwich scaffold. Structures of CAR
D1 alone and in complex with the Ad12 knob also have been solved
(24,
27). CAR D1 has a
-sandwich fold that is characteristic of Ig variable domains
(28,
29). The x-ray structure of
the Ad12 knob-CAR D1 binary complex indicates that no conformational
rearrangement occurs in either molecule upon complex formation and that over
50% of the Ad12 knob-CAR D1 interfacial contacts involve residues within the
knob AB loop, with the knob DE and EG loops also providing additional contact
residues (24). Mutational
analysis of the CAR binding activities of Ad5 and Ad12 knobs is consistent
with the Ad12 knob-CAR D1 x-ray structure, suggesting that the location of the
CAR D1-binding sites on knob domains from different serotypes is conserved
(24,
30). The trimeric knob domain
has three identical binding sites for CAR D1 that can be occupied
simultaneously, as observed in the x-ray structure of the binary complex and
in surface plasmon resonance experiments
(24,
31).
Alignment of the Ad12 knob amino acid sequence with sequences of knob
domains from other CAR-binding serotypes indicated that contact residues are
not well conserved. For example, of the 15 total contact residues in Ad12
knob, only six are identical in the Ad5 knob, whereas eight are identical in
the knob from Ad41L (32). This
extensive variation of contact residues implies that the mechanism of knob-CAR
interaction could differ significantly among adenovirus serotypes. Individual
contact residues at structurally analogous positions might differ considerably
in their relative contributions to binding affinity, as suggested recently in
a comparison of the interaction of recombinant knob domains from Ad5 and Ad9
with CAR D1 (31,
32). One question arising from
such studies is whether the relative positions of knob and CAR D1 in binary
complexes changes significantly with the variation of contact residues in the
knob component. This question could be addressed directly by solving crystal
structures of knob-CAR binary complexes, but so far we have been unable to
obtain diffracting crystals of binary complexes involving knob domains from
any adenovirus serotypes other than Ad12, for reasons not presently
understood. In this study, we investigated the structural basis for the
difference in affinity between Ad2 and Ad12 knob for CAR D1 by first
constructing homology models of Ad2 knob-CAR D1 binary complexes to predict
important differences in contact residues between the two serotypes. Ad12 knob
mutants were then constructed to test these predictions, both in functional
binding assays and ultimately in crystal structure determination of binary
complexes of the mutant knob proteins with CAR D1. Together, our results
suggest that mutation of individual contact residues, as might occur during
antigenic drift, in many cases only incrementally decreases affinity for the
receptor. Partial retention of binding affinity may enable mutant viruses to
successfully infect host cells and subsequently acquire secondary mutations
that either restore high affinity binding or shift binding specificity to a
novel receptor. Avidity effects resulting from the trivalent binding mechanism
of knob may further compensate for mutations that weaken binding affinity, as
suggested previously (31,
33,
34).
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EXPERIMENTAL PROCEDURES
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Protein Expression and PurificationRecombinant knob domain
from Ad12 fiber protein and the N-terminal domain of CAR (CAR D1) were
prepared as described previously
(15). The recombinant knob
domain from Ad2 fiber protein was prepared following the same protocol used
for Ad12 knob. Briefly, PCR products encoding the Ad2 and Ad12 fiber knob
domains and several flanking amino acids from the fiber shaft (Ad2 knob
residues 387582 and Ad12 knob residues 401587) were cloned
between the NdeI and BamHI restriction endonuclease cleavage
sites of vector pET15b (Novagen Inc., Madison, WI). The constructs were
transformed into strain BL21-DE3 (Novagen Inc., Madison, WI) for expression of
the hexahistidine-tagged knob proteins. All of the knob variants were
constructed by primer-directed PCR mutagenesis (mutagenic primers: Ad12 knob
P417S, GCAGTTTGGTGATGGGTCAGGAG; Ad12 knob S489Y, TCTATATCCCCAGTAAGCTTGAGGAA;
Ad2 knob S408P, TGCAGTTAGGAGGTGGGTCTGGG; and Ad2 knob Y477S,
TTCTAAAGTTCCAAGAATGTTTTTTAAGT) and cloned as above. The proteins were purified
via ion exchange and nickelnitriloacetic acid affinity chromatography (Qiagen
Inc., Valencia, CA), and the molecular weight of each construct was confirmed
by mass spectrometry. Each soluble expressed knob protein formed stable
trimers, as assessed by nondenaturing (native) polyacrylamide gel
electrophoresis and SDS-polyacrylamide gel electrophoresis of unheated protein
samples. PCR products encoding CAR D1 (CAR residues 21143, numbering
from the N-terminal signal peptide) with and without the S46C mutation were
cloned between the NcoI and XhoI restriction endonuclease
cleavage sites of pET15b. A stop codon was omitted from the reverse primer to
extend the CAR D1 C terminus with a 22-residue peptide encoded by vector
sequences. This peptide extension was found to increase folding of CAR D1 in
the Escherichia coli cytoplasm
(15). CAR D1 proteins were
expressed in strain BL21-DE3 and were purified by ion exchange chromatography.
CAR D1 mutation S46C was constructed by primer-directed mutagenesis, using the
mutagenic oligonucleotide primer GTCTTCGGGACAAAGCGTAAATTTG.
Labeling of CAR D1 S46CSerine 46 of CAR D1 was converted to
cysteine by primer-directed mutagenesis, and the resulting protein was
purified by ion exchange chromatography. CAR D1 S46C protein was covalently
labeled with 6-iodoacetamidefluorescein through the introduced reactive thiol
group of C46. The S46C protein (2.4 mg/ml) was reduced for 90 min in buffer A
(20 mM NaPO4, pH 8.0, 200 mM NaCl) containing
20 mM dithiothreitol. Excess dithiothreitol then was removed on a
Sephadex G-10 spin column equilibrated in buffer A. The reduced protein was
collected directly into a receiving tube containing 1.5 times molar excess of
6-iodoacetamide fluorescein, and the mixture was incubated for 4 h at 25
°C in the dark with end-over-end tumbling. Excess 6-iodoacetamide
fluorescein was removed by gel filtration on a Sephedex G-10 column (60
x 15 mm) in buffer A. The degree of labeling was determined
spectrophotometrically by measuring the absorbance of the labeled protein at
280 and 492 nm. Wild type CAR D1 control protein remained unlabeled after
treatment by this procedure, indicating that only the introduced cysteine
group of the S46C mutant was reactive.
Fluorescence Anisotropy ExperimentsEquilibrium binding
experiments were performed on a Tecan Ultra 384 plate reader (Tecan, Salzburg,
Austria) using flat bottomed, black, 96-well, untreated microplates (Nalge
Nunc Int., Naperville, IL). The reaction mixtures contained 5 nM of
fluorescein-labeled CAR D1 S46C in binding buffer (10 mM Tris-HCl,
pH 8.0, 0.1 mM EDTA, 0.0125% Nonidet P-40) with varying
concentrations of knob proteins (10 nM to 5 µM). The
total reaction volume was 300 µl, and the plates were incubated at 25
°C for 30 min. The fluorometer was set up with excitation and emission
wavelength filters of 485 and 535 nm, respectively, and with a fluorescein
dichroic mirror. The integration time was 40 µs, with 10 lamp
flashes/measurement. The gain and optimal Z-position were determined manually
using a control prior to the start of the experiment. The anisotropy values
were calculated as the ratio of the difference between vertical and horizontal
emission intensities (I// and I
)
normalized to the total intensity (Equation 1). Factor G is the ratio
of the sensitivities of the detection system for vertically and horizontally
polarized light.
 | (Eq. 1) |
The equilibrium binding curves were analyzed using Table Curve (SPSS Inc.,
Chicago, IL). Binding curves obtained for the knob-CAR interaction were fitted
to an A + B
AB model that describes single-site independent binding of
ligand to the receptor. This model assumes that complexes form with no
multivalent avidity effects, because both the receptor and the ligand are free
in solution during the analysis.
Competitive displacement studies were performed by titrating 5
nM of labeled CAR D1 S46C to
90% saturation with 650
nM knob domain from Ad2 fiber protein and then back titrating the
complexes with unlabeled wild type CAR D1 while measuring the decrease in
fluorescence anisotropy. The results of the concentration-dependent decrease
in anisotropy were fitted according to the equations of Huff et al.
(35). Because the
concentration of CAR D1 in the anisotropy assays (5 nM) was well
below the reported dissociation constant for CAR D1 homodimers (16
µM) (27), the
protein can be regarded as monomeric under the conditions used.
ModelingHomology modeling of Ad2 knob in complex with CAR
D1 was performed using Swiss PDB Viewer
(36). The Ad12 knob-CAR D1
binary complex was used as a template (PDB code 1KAC
[PDB]
), and Ad2 knob was
superimposed on the structure, 1.04 Å root mean square deviation for
-carbon atoms. Visual inspection of this model indicated potential
contacts at the interface of Ad2 knob and CAR D1 that are not observed in the
Ad12 knob-CAR D1 structure. The residues at the complex interface that were
not conserved between Ad2 and Ad12 knob were modeled in the context of Ad12
knob in all rotomer conformations. Positive and negative potential
interactions were scored using the Swiss PDB Viewer mutation tool.
CrystallizationCrystals of purified knob-CAR D1 complexes
were grown at room temperature by the sitting drop vapor diffusion method. All
of the reagents used were obtained from Hampton Research (Hampton Research,
Laguna Niguel, CA). Protein drops contained 2 µl of protein solution and 2
µl of reservoir solution, and the wells contained 500 µl of
crystallization buffer. Crystals of Ad12 knob P417S-CAR D1 were grown in 3.2
M ammonium sulfate in 100 mM HEPES, pH 7.0. The crystals
of the Ad12 knob S489Y-CAR D1 were grown in 0.8 M ammonium sulfate
in 100 mM HEPES, pH 7.0. The crystals were flash cooled at 99 K
with 50% ethylene glycol as a cryoprotectant.
Data Collection and Model RefinementIn each case, full data
sets were collected from single crystals using a 4 cell CCD on National
Synchrotron Light Source Beamline X25 at Brookhaven National Laboratory
(Upton, NY). The data were processed using the HKL Program suite
(37) as summarized in
Table II. The protein
coordinates of 1KAC
[PDB]
were subjected to rigid body refinement in CNS after a 5
Å region around the mutant site had been omitted. A cycle of torsion
angle refinement at 5000 K was performed prior to initial
Fo - Fc and 2Fo -
Fc electron density map calculation. The electron density
for the substituted amino acids could be assigned unambiguously. The final
refinement statistics are shown in Table
II. The coordinates for the P417S-CAR D1 and S489Y-CAR D1
structures were deposited in the PDB and assigned PDB codes 1P69 and 1P6A,
respectively.
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RESULTS
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Fluorescence Anisotropy Assay and Design of Knob
VariantsThe residues involved in the recognition of CAR D1 are not
strictly conserved within the knob domains of fiber proteins from different
adenovirus serotypes (Fig. 1).
To assess the effects of this variation on binding affinity, we developed a
fluorescence anisotropy-based assay that measures interaction of knob and CAR
D1 in solution. This assay limits multivalent avidity effects that can arise
when either knob or CAR D1 is immobilized on a two-dimensional surface,
because it measures the affinity of individual binding sites on the trivalent
knob molecule. For this assay, a reactive sulfhydryl group was introduced into
CAR D1 to permit site-specific labeling with fluorescein. Serine 46 was chosen
for conversion to cysteine based on its surface exposure and its location
outside of the knob-binding surface of CAR D1
(Fig. 2C).
Spectroscopic analysis showed that the CAR D1 S46C mutant protein was
quantitatively labeled after reaction with 6-iodoacetamide fluorescein,
whereas no incorporation of fluorescein was detected in control reactions
containing wild type CAR D1 protein (data not shown). Formation of knob-CAR D1
complexes was monitored by the increase in fluorescence anisotropy upon
incubation of fluorescein-labeled CAR D1 S46C with increasing, subsaturating
concentrations of knob protein (Fig.
2A). To determine whether the fluorescein label
influenced the interaction of CAR D1 with the knob proteins, a competitive
displacement experiment was performed. Fluorescein-labeled CAR D1 S46C was
titrated to
90% saturation with Ad2 knob protein, and the complexes were
then back titrated with wild type unlabeled CAR D1
(Fig. 2B). The data
were fitted to a displacement curve to determine the inhibition constant
(Ki). The dissociation constant
(Kd) of the forward titration and the
Ki of the back titration were equivalent to
within 3% error, indicating that the labeled CAR D1 S46C protein retained
binding activity similar to that of wild type CAR D1.

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FIG. 1. Sequence alignment of Ad2 and Ad12 knob. Sequences of Ad2 knob
residues 399582 and Ad12 knob residues 408587 were aligned using
ClustalX (42). Conserved
residues are indicated with a asterisk, and similar residues are
indicated with one or two dots. Loop regions are
underlined and named. Highlighted in red are residues of
Ad12 knob that contact CAR D1 in the Ad12 knob-CAR D1 crystal structure.
Residues mutated in this study are boxed in blue. Six of the
15 Ad12 knob residues involved in contacts with CAR D1 are strictly conserved
in Ad2 knob. Of the remaining nine Ad12 knob contact residues, seven
correspond to conservative substitutions (indicated by dots), and two
correspond to nonconservative substitutions in Ad2 knob.
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FIG. 2. Analysis of knob-CAR D1 interactions using equilibrium fluorescence
anisotropy. A, equilibrium binding curve of Ad2 knob with labeled
CAR D1 S46C. B, displacement of labeled CARD1 S46C from Ad2 knob by
wild type CAR D1. Labeled CAR D1 S46C was back titrated from a near saturating
concentration of Ad2 knob using an increasing amount of wild type CAR D1.
C, stereo view of CAR D1 mutant S46C in ribbon representation. The
side chain of residue 46 is highlighted in yellow. Residues that
interact directly with Ad12 knob are shown in ball and stick
representation.
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Using this anisotropy assay, we measured the binding affinities of two
different serotypes of adenovirus knob, Ad2 and Ad12, toward CAR D1.
Equilibrium binding affinities were derived from binding isotherms, as
reported in Table I. Ad2 knob
bound to CAR D1 with a Kd of 35 nM,
whereas Ad12 knob bound to CAR D1 with a Kd of
295 nM. To investigate the basis for this 8-fold difference in
affinity, we constructed a homology model (rigid body interaction) of the Ad2
knob-CAR D1 complex (Fig. 3).
Because the main chain conformations of Ad2 and Ad12 knob are similar
(
-carbon atoms have root mean square deviation of 1.04 E), Ad2 knob was
built into the homology model in the same relative orientation as that of Ad12
knob in the crystallographically determined Ad12 knob-CAR D1 complex. No
steric clashes were observed at the binding interface, suggesting that the
homology model reasonably approximated the actual structure of the Ad2
knob-CAR D1 complex.
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TABLE I Equilibrium binding affinities of adenovirus knob domains for CAR D1
obtained through fluorescence anisotropy measurements The results are the
means ± S.D. of triplicate measurements.
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FIG. 3. Homology model of Ad2 knob bound to CAR D1. The x-ray structure of
Ad2 knob was overlaid onto Ad12 knob in the x-ray structure of the Ad12
knob-CAR D1 complex. For simplicity, only one monomer of the knob trimer is
shown. The surface of CAR D1 (space-filling model on left) is colored
blue. Superimposed ribbon structures of Ad2 knob and Ad12 knob are
colored cyan and green, respectively. Side chain residues of
Ad12 knob contacting CAR D1 are shown in gray except for side chains
of Ser489 and Pro417, which are shown in red.
Ad2 knob Ser408 and Tyr477 side chains are colored
yellow.
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Six contact residues of Ad12 knob are strictly conserved in Ad2 knob, and
all of these appear to contact CAR D1 in the homology model of Ad2 knob-CAR
D1. Seven contact residues of Ad12 knob are conservatively substituted in Ad2
knob, and three of these Ad2 knob residues (Asn482,
Thr486, and Thr507) appear to contact CAR D1, whereas
three others (Ser442, Lys475, and Gln508) are
more distant from CAR D1 and thus may contribute less to binding affinity. The
final Ad2 knob residue in this group, Ser408, appeared to form an
additional hydrogen bond with CAR D1 that is not observed in the Ad12 knob-CAR
D1 complex. The remaining two contact residues of Ad12 knob (Leu426
and Glu523) correspond to nonconservative substitutions in Ad2
knob. Ad12 knob residue Leu426 forms a main chain hydrogen bond
with CAR D1, and the corresponding Ad2 knob residue, Asn417, also
is within hydrogen bonding distance from CAR D1 in the homology model. Ad12
knob Glu523 forms hydrophobic contacts with CAR D1, but the
corresponding Ad2 knob residue (Thr511) is too distant from CAR D1
to make similar contacts. However, small rearrangements of the EG loop
compared with its position in the model potentially could enable
Thr511 to contact CAR D1. In summary, homology modeling suggested
that 4 of the 15 residues of Ad2 knob that correspond to contact residues in
Ad12 knob may not be oriented favorably to contact CAR D1. Consequently, we
searched the homology model further for additional potential contact residues
that could account for the greater affinity of Ad2 knob for CAR D1.
It was observed that one Ad2 knob residue in particular, Tyr477,
was within hydrogen bonding distance of CAR D1 and that its side chain was
able to make additional contacts at the complex interface. Tyr477
is positioned within a region that forms a cavity in the Ad12 knob-CAR D1
complex (Fig. 3). Modeling of
tyrosine at the corresponding position in Ad12 knob, Ser489,
indicated that a tyrosine residue at this position could be accommodated in
the Ad12 knob-CAR D1 interface and could form two additional hydrogen bonds
with CAR D1. Therefore, the overall results of homology modeling suggested
that the increased affinity of Ad2 knob for CAR D1 could result from
additional contacts contributed by Ad2 knob residues Ser408 and
Tyr477.
To test the importance of Ser408 and Tyr477 in the
interaction of Ad2 knob with CAR D1, these residues were mutated to the
corresponding residues in Ad12 knob, proline and serine, respectively, and the
interaction of the resulting Ad2 knob mutants S408P and Y447S with CAR D1 was
characterized in the fluorescence anisotropy assay
(Fig. 4). The affinity of Ad2
knob S408P was decreased by approximately a factor of 2, whereas the affinity
of Ad2 knob Y477S was decreased
100-fold
(Table I). The converse mutants
also were constructed to determine whether additional contacts contributed by
these residues would increase the binding affinity of Ad12 knob. Equilibrium
binding studies showed that the resulting Ad12 knob mutants P417S and S489Y
both had increased affinity for CAR D1
(Table I and
Fig. 4), consistent with the
model that the side chains of both mutant residues make positive contacts with
CAR D1. A double mutant, introducing both point mutations into Ad12 knob,
further increased the binding affinity to 28 nM, greater than the
observed affinity of wild type Ad2 knob for CAR D1
(Fig. 4).

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FIG. 4. Anisotropy measurements of fluorescein-labeled S46C-CAR D1 with
adenovirus knob variants. The results are the means of triplicate
measurements. The error bars represent the standard deviations.
WT, wild type.
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X-ray Structures of Ad12 Knob Mutants in a Binary Complex with CAR
D1To further investigate the interactions that contribute to the
increased binding affinity of the Ad12 knob mutants P417S and S489Y, x-ray
structures of the two mutant proteins in binary complexes with CAR D1 were
solved to
3 Å resolution (Table
II). Data sets were essentially isomorphous to the native data
sets, resulting in corresponding structures that are very similar to the
native structure. Backbone atoms of wild type Ad12 knob-CAR D1 and Ad12 knob
P417S-CAR D1 were superimposable with a root mean square deviation of 0.1
Å, indicating that the overall main chain structures are identical and
that CAR D1 was bound in the same conformation and orientation in both the
mutant and wild type complexes. Backbone atoms of wild type Ad12 knob-CAR D1
and Ad12 knob S489Y-CAR D1 also were superimposable with a root mean square
deviation of 0.1 Å, again indicating identical conformations and
orientation of bound CAR D1. The
-hydroxyl group of the serine residue
introduced into the AB loop of Ad12 knob (P417S) is within hydrogen bonding
distance of the O
1 atom of CAR D1 residue Glu56
(Fig. 5b) and thus
potentially could form an additional hydrogen bond within the knob-CAR D1
interface. No other conformational changes, with the exception of a rotation
of the side chain of CAR D1 residue Asp54 at the C
atom, were
observed compared with the structure of the wild type binary complex,
suggesting that the increase in affinity of this mutant may be a direct
consequence of the serine residue at position 417. The crystal structure of
the Ad12 knob S489Y-CAR D1 complex indicated that the extended length of the
tyrosine residue side chain at residue 489 increased its potential to form
additional hydrogen bonds with CAR D1 that cannot form by the corresponding
serine residue in wild type Ad12 knob. The -OH group of knob residue
Tyr489 was within hydrogen bonding distance of the backbone oxygen
and nitrogen atoms of CAR D1 residues Pro52 and Ala125
(Fig. 6b). Rotation of
the side chain of CAR D1 residue Asp54 again was the only observed
change in conformation as compared with the wild type Ad12 knob-CAR D1
structure. The substantial increase in affinity of Ad12 knob mutant S489Y for
CAR D1 therefore likely results from formation of two additional hydrogen
bonds and burial of the tyrosine aromatic side chain.

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FIG. 5. Comparison of crystal structures of wild type Ad12 knob-CAR D1 and Ad12
knob P417S-CAR D1. a, stereo figure showing part of the wild type
Ad12 knob-CAR D1 interface including CAR D1 residues I55-E56-W57
(cyan) and Ad12 knob residues P416-P417-P418 (yellow); no
hydrogen bonds are formed at this interface. b, stereo figure of same
view of the interface between Ad12 knob mutant P417S (blue) and CAR
D1 (red); note the novel hydrogen bond formed between
Ser417 of knob and Glu56 of CAR D1.
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FIG. 6. Comparison of crystal structures of wild type Ad12 knob-CAR D1 and Ad12
knob S489Y-CAR D1. a, stereo figure showing part of the wild type
Ad12 knob-CAR D1 interface including CAR D1 residues
Gly51-Pro52-Leu53 and
Lys124-Ala125-Pro126 (cyan) and Ad12
knob residues Ala488-Ser489-Trp490
(yellow); no atoms are within hydrogen bonding distance at this
interface. b, same view of the interface between Ad12 knob mutant
S489Y (blue) and CAR D1 (red). Note that Tyr489
of knob makes two novel hydrogen bonds with Pro52 and
Ala125 of CAR D1.
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DISCUSSION
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Evolution of adenoviruses under immunoselective pressure has resulted in
antigenic drift of surface residues, including those within the
receptor-binding sites of the fiber protein knob domain. Despite this high
degree of surface variation, many adenovirus serotypes attach to cells through
interaction with CAR, a membrane glycoprotein that also serves as a cellular
receptor for group B coxsackieviruses. Investigation of the interaction of
fiber proteins from different adenovirus serotypes with CAR D1 therefore
presents an opportunity to estimate the range of solutions to the molecular
recognition problem that occur in a natural system. With the exception of Ad12
knob, it has so far not been possible to directly view the interaction of
knobs from other adenovirus serotypes with CAR by x-ray crystallography. Here
we circumvented this problem by using the x-ray structure of Ad12 knob-CAR D1
binary complex as a template to model contact residues on the surface of other
knob serotypes and predict how binding affinity and specificity might be
affected by variation of contact residues.
Ad2 knob was chosen for this initial modeling study because it binds to CAR
D1 with significantly greater affinity than does Ad12 knob
(Table I). Equilibrium binding
studies were performed using fluorescence anisotropy measurements of CAR D1
labeled with fluorescein at a specific site. Affinity values calculated from
the anisotropy measurements were lower (weaker) than have been previously
observed in other in vitro assays, such as surface plasmon resonance
(31,
32). These differences in
affinity probably can be attributed to the absence of avidity effects in
complex formation. In binding assays such as surface plasmon resonance, either
the receptor or the ligand is immobilized, thereby limiting the association
event to a two-dimensional surface. By contrast in the fluorescence anisotropy
assay used here, both interacting species are in solution, and the affinity
values therefore reflect individual binding events. Although the absolute
affinity values obtained by fluorescence anisotropy probably underestimate the
actual overall affinity of knob for membrane-associated CAR, the values
obtained by this method are useful for characterizing the contribution of
individual amino acid residues within the knob-CAR interface. Equilibrium
binding affinity measurements using the fluorescence anisotropy assay showed
that Ad2 knob bound CAR D1 with an 8-fold greater affinity compared with Ad12
knob (Table I). The increased
affinity of Ad2 knob indicates that its receptor-binding sites overall make
more favorable contacts with CAR D1 than do the binding sites of Ad12 knob.
Binding site fitness could be improved either by increasing the number of
favorable contacts or by eliminating contacts that interfere with stable
binding.
The homology model of the Ad2 knob-CAR D1 complex predicted that two
residues of Ad2 knob, Ser408 and Tyr477, could
contribute additional hydrogen bonds and other contacts that may account for
the greater affinity of Ad2 knob for CAR D1
(Fig. 3). These predictions
were well supported by mutagenesis studies, where substitution of serine and
tyrosine for the corresponding residues in Ad12 knob increased its affinity
for CAR D1, whereas the converse mutations decreased the affinity of Ad2 knob
for CAR D1. Prior studies using kinetic-based measurements indicated that Ad12
knob has a slower on-rate but similar off-rate compared with Ad2 and Ad5 knobs
(31,
32). Although kinetic
parameters cannot be determined from the equilibrium binding assay used here,
it is likely that the P417S and S489Y mutations increase the association rate
of Ad12 knob through the formation of more favorable interfacial contacts. The
P417S and S489Y mutations are located in the Ad12 knob AB and DE loops,
respectively, which is consistent with the earlier conclusion that individual
residues within these two loops make substantial contributions to binding
affinity (24,
30). Importantly, whereas
several earlier studies have predicted contact residues in knob serotypes
based on amino acid sequence alignments with Ad12 knob
(14,
24,
30,
32,
38), here through the use of
homology modeling we were able to identify a novel contact residue in Ad2 knob
(Y477) that does not have a functionally equivalent counterpart in Ad12 knob.
Substitution of tyrosine for the positionally equivalent residue in Ad12
(Ser489) resulted in an 8-fold increase in binding affinity.
To directly investigate the contribution of individual residues to binding
affinity, we crystallized and solved the structures of two Ad12 knob mutants,
P417S and S489Y, in complex with CAR D1. No changes in the relative positions
of knob and CAR D1 components were detected in the resulting 3 Å
resolution structures, as compared with the wild type complex, and no
significant perturbations of the Ad12 knob structure were detected for either
mutant. The structures support predictions from modeling studies that each
substituted residue forms additional hydrogen bonds with CAR D1. Homology
modeling also predicted that the protruding aromatic side chain of Ad2 knob
residue Tyr477 would reduce the size of a cavity that forms in the
Ad12 knob-CAR D1 interface
(24), possibly excluding some
nonstructural water molecules that become trapped in the Ad12 knob-CAR D1
complex. Burial of an aromatic side chain can play a critical role in
stabilizing protein-protein interactions. For example, in the interaction of
human immunodeficiency virus with its cellular receptor CD4, insertion of CD4
residue F43 into a cavity on the surface of gp120 accounts for 23% of
interatomic contacts at the interface
(39). The 100-fold decrease in
Ad2 knob binding affinity resulting from mutation Y477S indicates that
Tyr477 also makes a large contribution to binding affinity,
although the precise role of the residue cannot be fully understood without
determining a high resolution structure of the Ad2 knob-CAR D1 complex. The
relatively smaller effect of tyrosine at the equivalent position in Ad12 knob
(mutant S489Y) suggests that the contribution of individual contact residues
to binding affinity may be context-dependent. At 3 Å resolution it was
not possible to determine the impact of the S489Y mutation on the size and
solvent content of interfacial cavities.
The high degree of variation in contact residues among CAR-binding
serotypes and the observed tight range of binding affinities (variations over
a
10-fold range have been measured by surface plasmon resonance
(31,
32) with the exception of Ad9
knob (32)) suggest that the
knob domain architecture may be specialized to minimize the impact of
mutations on binding site structure and function. The receptor binding sites
of knob consist of surface loops that are supported by an unusually stable,
trimeric scaffold, and in this regard have a similar architecture to the
antigen binding sites of antibodies. Although the range of molecular targets
recognized by knob domains of natural adenoviruses appears limited to a small
number of cellular receptors, this narrow range undoubtedly reflects the
strong selective pressure on the virus to retain sufficient binding affinity
to successfully infect host cells. It is probable that the natural evolution
of binding specificity is a multi-step process where the loss of affinity for
the initial receptor and gain of affinity for a novel receptor occur
simultaneously. Multivalent binding may compensate for relatively weak or low
affinity binding at individual sites, enabling virus to bind tightly enough to
infect host cells (31). In
addition, certain adenovirus serotypes have two independent fiber genes
(40), which may have provided
an alternate means for independent evolution of novel binding specificity.
Changes in fiber protein binding specificity during virus evolution likely
contributed to the overall success of adenoviruses as human and animal
pathogens. The utility of adenovirus-based vectors for use in gene therapy or
as recombinant virus vaccines also might be enhanced by rational modification
of fiber protein binding specificity. The availability of bacterial expression
systems for recombinant knob domain
(5) and of methods for
structure-guided mutagenesis and directed protein evolution
(41) now can be exploited to
investigate the potential of the knob scaffold to interact with a broad range
of molecular targets.
 |
FOOTNOTES
|
---|
* This work was supported by Grant R01-AI36251 from the United States Public
Health Service and by a grant from the United States Department of Energy
Office of Biological and Environmental Research under Contract
DE-AC02-98CH10886. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be hereby
marked "advertisement" in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact. 
Present address: Dept. of Biological Sciences, Biophysics Section, Blackett
Laboratory, Imperial College London, Prince Consort Road, London SW7 2BW,
UK. 
To whom correspondence should be addressed: Biology Dept., Brookhaven National
Laboratory, Upton, NY 11973. Tel.: 631-344-3350; Fax: 631-344-3407; E-mail:
freimuth{at}bnl.gov.
1 The abbreviations used are: CAR, coxsackievirus and adenovirus receptor;
D1, domain 1; Ad, adenovirus serotype; PDB, Protein Data Bank. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Karen Springer for excellent technical assistance.
 |
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