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INTRODUCTION |
Adenoviruses are responsible for a variety of respiratory,
gastroenteric, and ocular infections (1). They are DNA viruses that
predominantly infect mammals and birds. In humans 51 types have been
identified and grouped into six distinct classes, A to F. Adenoviruses
form icosahedral particles with 240 copies of the trimeric hexon
protein arranged on the planes and a penton complex at each of the 12 vertices. The penton complex consists of a pentameric base, implicated
in virus internalization (2), and an externally protruding trimeric
fiber. The fiber protein is responsible for the initial attachment to
the host cell, through its C-terminal head domain (3). The atomic
structures of type 2 and type 5 fiber heads are known (4, 5);
furthermore, the fiber shaft has been shown to contain a novel triple
-spiral fold (6).
The receptor of most adenovirus types has been identified to be a cell
surface protein named coxsackievirus and adenovirus receptor
(CAR)1 (7, 8). CAR has been
shown to be the receptor for subgroup A, C, D, E, and F virus fibers
(9) but not for subgroup B (to which, for example, adenovirus type 3 and 7 belong). Short fibers from group F also do not bind CAR. The
receptor-binding site on the adenovirus fiber head has recently been
described based on comparative sequence analysis of CAR-binding and
non-CAR-binding fiber head domains (4, 10) and mutagenesis studies (10, 11). The structure of adenovirus type 12 fiber head complexed with the
CAR N-terminal Ig D1 domain, which is necessary and sufficient for adenovirus attachment (12), is also known (11). Together, the data
show that the primary determinant for CAR binding on the fiber head is
the AB loop (for a definition of
-strands and loops of the fiber
head, see Ref. 5). The AB loops are located on the sides of the head
trimer close to an adjacent monomer. Binding of CAR to the fiber does
not appear to lead directly to intracellular signaling events important
for adenovirus infection because the intracellular domain plus
transmembrane helix of CAR can be replaced by another membrane anchor
with retention of CAR-mediated adenovirus infection (13). CAR has been
proposed to function as a homophilic cell adhesion molecule (14), a
proposition supported by the observation that the CAR D1 domain forms
homodimers in solution and in the crystalline state (15).
Here we describe surface plasmon resonance (SPR) binding studies of
adenovirus fiber to the D1 domain of CAR, using bacterially expressed
proteins. We have determined the relevant association and dissociation
constants of this interaction and report an avidity-based mechanism by
which the adenovirus fiber binds to three receptor molecules
immobilized on a surface. We propose that adenovirus fiber and other
multimeric ligands bind their receptors on the cell surface in an
analogous way.
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EXPERIMENTAL PROCEDURES |
Construction of Expression Vectors--
For the expression of
the adenovirus type 2 fiber head, the plasmid pT7.Ad2fib388-582 was
constructed. A DNA fragment coding for residues 388-582 was obtained
by polymerase chain reaction using pTaq (3) as a template and
cloned into the vector pPMD (16), from which the
NcoI-BamHI insert was removed (pPMD is a variant
of pT7-7, a low copy number vector employing the T7 RNA polymerase
system). For the construction of pAB3.CAR15-140H, a DNA fragment
encoding residues 15-140 was obtained by polymerase chain reaction
using the plasmid pcDNA1-CAR (7) as a template and cloned into the
expression vector pAB3 (17), which permits expression under control of
the lac promotor-operator and targeting to the bacterial
periplasm by means of a pectate lyase leader peptide. The resulting
protein contains a C-terminal metal-binding tag.
Protein Expression and Purification--
The adenovirus type 2 fiber head was expressed in Escherichia coli strain
JM109(DE3) (Promega, Charbonnières, France). Bacteria transformed
with pT7.Ad2fib388-582 were grown in LB-Ap medium (10 g/liter Bacto
tryptone, 5 g/liter yeast extract, 7.5 g/liter sodium chloride, 100 mg/liter ampicillin) at 37 °C to an optical density of 0.5 at 600 nm. They were then cooled to 22 °C, 0.5 mM
isopropyl-
-D-thiogalactopyranoside was added, and growth
was continued for 16 h at 22 °C. Cells from 5 liters of culture
were resuspended in 100 ml of 50 mM Tris-HCl, pH 8.0, 25 mM sodium chloride, 1 mM dithiothreitol
containing protease inhibitors (CompleteTM; Roche Molecular
Biochemicals) and lyzed using a French press. Insoluble material was
removed by centrifugation, nucleic acids were precipitated by adding
1% (w/v) streptomycin sulfate, and 70 ml of a saturated solution of
ammonium sulfate was added to the supernatant. After centrifugation,
the supernatant was loaded onto 75 ml of phenyl-Sepharose FF high
subcolumn (Amersham Pharmacia Biotech) equilibrated with PE buffer (25 mM sodium dihydrogen phosphate, 25 mM disodium
hydrogen phosphate, 1 mM EDTA, pH 6.8) containing
1.5 M ammonium sulfate. Elution was with a gradient of
1.5-0 M ammonium sulfate; the protein eluted at about 0.6 M. Fractions containing the desired protein were dialyzed
against 10 mM Tris-HCl, pH 8.5, 1 mM EDTA and
loaded onto a Q10 anion exchange column (Bio-Rad) equilibrated in the
same buffer. This column was eluted with a gradient of 0-200
mM sodium chloride; the protein eluted at about 30 mM. Fractions containing the desired protein were pooled,
brought to 50 mM phosphate, pH 6.8, and 1.5 M
ammonium sulfate by adding concentrated stock solutions, and loaded
onto a phenyl superose 10/10 column (Amersham Pharmacia Biotech)
equilibrated in PE buffer containing 1.5 M ammonium
sulfate. Elution was with a gradient of 1.5-0 M ammonium
sulfate; the protein eluted at about 1.3 M. Fractions
containing pure protein as judged by SDS-polyacrylamide gel
electrophoresis (PAGE) electrophoresis were pooled and transferred to
HN buffer (10 mM HEPES, 150 mM sodium
chloride, pH 7.4, with sodium hydroxide) by repeated dilution and
concentration using Centricon Plus-20 devices with a nominal molecular
mass cut-off of 10 kDa (Millipore, St. Quentin en Yveline, France). An adenovirus fiber protein containing part of the shaft region (residues 319-592; Ref. 6) was similarly expressed and purified
using the plasmid pT7.Ad2fib319-582.
The D1 domain of CAR was expressed in E. coli with a
12-residue C-terminal extension (GAPAAAHHHHHH) to enable purification by metal-chelating chromatography. The resulting protein will be
referred to as CAR D1. Cultures of E. coli strain XL1Blue
(Stratagene, La Jolla, CA) transformed with pAB3.CAR15-140H were grown
in LB-Ap medium at 25 °C to an optical density of 0.5 at 600 nm.
Expression was induced with 0.2 mM
isopropyl-
-D-thiogalactopyranoside, and growth continued
for 10 h at 25 °C. Cells from 5 liters of culture were washed
with 150 ml of phosphate-buffered saline solution (pH 7.4). Washed
cells were resuspended in 150 ml of 200 mM Tris-HCl, pH
8.0, 0.5 mM EDTA, 0.5 M sucrose, centrifuged as
before, and resuspended in 150 ml of distilled water to release the
periplasmic fraction. After a 20 min of incubation, cell debris were
removed by centrifugation, and protease inhibitors
(CompleteTM EDTA-free; Roche Molecular Biochemicals) were
added. The protein was bound to 2 ml of nickel-nitrilotriacetic acid
slurry (Qiagen, Courtaboeuf, France) following the instructions
supplied and eluted stepwise with imidazole solutions of increasing
concentrations at pH 7.5; the protein eluted between 30 and 200 mM imidazole. Further purification was using a phenyl
superose 10/10 column as described for the fiber head protein. The
protein eluted at about 1.1 M ammonium sulfate. Fractions
containing pure protein as judged by SDS-PAGE electrophoresis were
transferred to HN buffer as described above. A protein comprising
residues 15-140 without a His tag was also expressed in E. coli and purified (15).
The molar extinction coefficients of the expressed proteins were
determined by UV spectroscopy combined with amino acid analysis. Molar
extinction coefficients at 280 nm of 35.5 × 103
M
1 cm
2 for the adenovirus type
2 fiber head (residues 388-581), 38.2 × 103
M
1 cm
2 for adenovirus type 2 head plus part of shaft fiber protein (residues 319-582), 18.1 × 103 M
1 cm
2 for
His-tagged CAR D1, and 18.5 × 103
M
1 cm
2 for non-His-tagged CAR
D1 were obtained. Protein concentrations were then determined by UV
spectroscopy alone. Dynamic light scattering measurements were
performed with DynaPro-MS800 dynamic light scattering/molecular sizing
instrument (Protein Solutions Ltd., Charlottesville, VA).
SPR Measurements and Analysis--
Surface activation, protein
immobilization, and binding assays were carried out using an upgraded
Biacore 1000 SPR measurement apparatus (Biacore, St. Quentin en
Yveline, France). Flow cells of a Biacore B1 sensor chip were activated
with a cross-linking mixture consisting of 50 µl of 0.2 M
N-ethyl-N'-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride and 0.05 M N-hydroxysuccinimide,
after which adenovirus type 2 fiber head (25 µg/ml in 10 mM sodium acetate, pH 5) or CAR D1 (20 µg/ml in 10 mM sodium acetate, pH 5.0) was injected. Different amounts
of immobilized protein were obtained by varying the injected volume.
Remaining activated groups were blocked with 50 µl of 1 M
ethanolamine, pH 8.5. A third flow cell was activated with the
cross-linking mixture and immediately blocked with ethanolamine to
serve as a negative control. No significant binding of fiber head or
CAR D1 to the negative control flow cell was observed (not shown).
All binding assays were carried out at 25 °C, and HBS buffer (10 mM HEPES, 150 mM sodium chloride, 3 mM EDTA, 0.005% P20 detergent, pH 7.4) was used as running
buffer. CAR D1 was diluted in running buffer, and different
concentrations were injected over the fiber head surface for 5 min each
to study the association and equilibrium phase. The surface was then
washed with HBS buffer for 15 min to study the dissociation phase. The
fiber head-CAR D1 complex fully dissociated during that period of time,
and regeneration steps were not necessary. In a second series of
experiments, fiber head was injected over a CAR D1 surface. After each
injection, the surface was regenerated with a 2-min pulse of 10 mM hydrochloric acid.
Maximum stoichiometries of binding were determined by comparing the
increase in resonance units (RU) during the immobilization procedure
with the maximal increase in RU during the binding phase. The increase
in RU depends linearly on the increase in bulk refractive index, and
the specific refractive index increment is closely similar for a wide
range of proteins independent of amino acid composition. The increase
in RU can thus be regarded as depending linearly on the mass of the
molecules or complexes attached to the surface, within the limits of
measurement. The stoichiometry can then be calculated by dividing the
fractional increase in RU by the fractional molecular mass of the
concerned species.
Where possible, equilibrium data were extracted from the sensorgram at
the end of each injection and used to calculate the equilibrium
dissociation constant independently of the kinetic analysis. Kinetic
constants (kon and koff)
were derived from the association and dissociation curves of the
sensorgrams either by linear transformation of the primary data or
nonlinear fitting of the sensorgrams and by numerical integration
(global fitting) of the data to different interaction models, using the
Biacore BIAevaluation 3.1 software supplied with the apparatus. Binding curves obtained when CAR D1 was allowed to bind to immobilized fiber
head were fitted to a simple A + B
AB model provided with the software. When fiber head bound
to immobilized CAR D1, we used the rate Equations 1-3 to fit our data,
which describe "trivalent binding" (one trimeric fiber head can
bind up to three monomeric CAR D1 molecules), according to Equations
4-7, where A is the trivalent fiber head, B is
the CAR D1 molecule, kon1,
kon2, and kon3 are the
association rate constants of Equations 1, 2, and 3, respectively, and
koff1, koff2, and
koff3 are the dissociation rate constants of
Equations 1, 2, and 3, respectively.
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(Eq. 1)
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(Eq. 2)
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(Eq. 3)
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(Eq. 4)
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(Eq. 5)
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(Eq. 6)
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(Eq. 7)
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RESULTS |
Expression in Bacteria and Purification of Recombinant
Proteins--
The adenovirus type 2 fiber head protein containing
residues 388-582 of the coding sequence, henceforth to be referred to as "fiber head," was expressed in E. coli and purified
by a combination of hydrophobic interaction and anion exchange
chromatography. The preparation yielded 3-5 mg of purified
protein/liter of bacterial culture. N-terminal sequence analysis gave
the correct sequence (AITIGNKNDD) and showed that the introduced
methionine was post-translationally removed. The same protein was
previously expressed in insect cells (3). We also expressed a construct
containing the head domain plus part of the shaft domain containing
residues 319-582 (6).
Based on comparison with the extracellular domain of CD4, of which the
structure is known (18), we defined the N-terminal Ig domain of CAR
(D1) as containing residues 15-140 of the CAR coding sequence. Residue
15 in this numbering was thought to be the N-terminal residue of the
mature CAR protein; residues 1-14 were thought to be a signal sequence
that is removed after targeting to the cell membrane (7). It was later
shown that the mature CAR protein starts at residue 20 (19). We
expressed the CAR D1 domain with a C-terminal His tag in E. coli using an expression system in which the protein is exported
to the periplasm. Purification was by metal-chelating and hydrophobic
interaction chromatography (see "Experimental Procedures") to
homogeneity as judged by SDS-PAGE. The preparation of CAR D1 yielded
2-3 mg of pure protein/liter of bacterial culture. N-terminal sequence
analysis gave the sequence DFARSLSITT, which confirmed the identity of
the expressed protein and the removal of the leader sequence. We also
expressed and purified a nontagged version of CAR D1 (15). CAR D1 has
also been successfully expressed in the cytoplasm of E. coli
by Freimuth et al. (12).
Aggregation State of the Bacterially Expressed Proteins--
The
natural adenovirus type 2 fiber is a trimer of exceptional stability.
It is resistant to incubation with SDS at moderate temperatures and
only dissociates into monomers if incubated with SDS at higher
temperatures (20, 21). Type 2 fiber head containing residues 388-582
expressed in baculovirus-infected insect cells has been shown to be
trimeric by covalent cross-linking (3), crystallographic analysis (4),
and dynamic light scattering. Our bacterially expressed fiber head
protein (also containing residues 388-582) shows the same resistance
to SDS as fiber protein isolated from adenovirus and as the fiber head
expressed in baculovirus-infected insect cells. It is also trimeric
when analyzed by gel filtration of SDS-PAGE without prior boiling of
the sample and monomeric when the sample is heated before applying it
to the gel (Fig. 1, B and
C). Furthermore, it forms crystals identical to the protein produced in baculovirus-infected insect
cells.2

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Fig. 1.
SDS-PAGE analysis of the CAR D1 domain and
the adenovirus type 2 fiber head. Proteins were analyzed on a gel
containing 12.5% acrylamide and 0.1% SDS. A, His-tagged
CAR D1. B, fiber head with prior boiling of the sample.
C, fiber head without prior boiling of the sample. Molecular
size markers were loaded to the left of lane A
and to the right of lane B; their sizes are
indicated on the left in kDa.
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The bacterially expressed head plus shaft construct is also trimeric in
SDS at moderate temperature or when analyzed by gel filtration or
dynamic light scattering. The mass obtained by dynamic light scattering
for the head plus shaft construct was 89.0 kDa, with no discernible
polydispersity, closely corresponding to a value of 85.7 kDa expected
for a trimer. We can therefore safely assume that both proteins are
trimeric under the nondenaturing conditions of our surface plasmon
resonance assays.
The aggregation state of non-His-tagged CAR D1 has been studied
previously (15). It was found to dimerize in the crystal and also in
solution. In solution, a dissociation constant of 16 µM
could be measured by equilibrium centrifugation. We also performed
equilibrium centrifugation experiments with His-tagged CAR D1 and
obtained a dissociation constant of 25 ± 10 µM.
Within error, this value overlaps the one obtained for non-His-tagged CAR D1. Because our surface plasmon resonance assays contained CAR D1
at nanomolar concentrations, the protein can be regarded as monomeric
under these conditions.
CAR D1 Binding to Immobilized Adenovirus Type 2 Fiber Head--
To
determine conditions suitable for kinetic analysis, we prepared
surfaces with different amounts of immobilized adenovirus type 2 fiber
head. CAR D1 was injected onto these surfaces, using different flow
rates. Because of the fast dissociation, it was not necessary to
include a regeneration step after each CAR injection, and a 15-min
injection of running buffer was enough to completely remove CAR D1 from
the immobilized fiber head. The primary data were analyzed by linear
transformation ("linearization," see Ref. 22) using a simple
A + B
AB model to give the kinetic
parameters reported in Table I.
Increasing the flow rate from 10 to 50 µl/min over a surface
containing 1200 RU of fiber head did not change the kinetic parameters
(Table I), indicating that the binding reaction was not limited by mass
transport effects. We also investigated surfaces on which 600 or 1500 RU of fiber head were immobilized. There was no significant dependence
on the surface density of the kinetic parameters of CAR D1 binding to
immobilized fiber head (Table I). The binding of CAR D1 molecule to
immobilized fiber head can thus be characterized by an on rate constant
kon = 2.7 ± 0.3 × 105
M
1 s
1 and a dissociation rate
constant koff = 6.7 ± 0.6 × 10
3 s
1 (errors are standard deviations from
the five different experiments in Table I), leading to an average
dissociation constant of 24.9 ± 1.2 nM. Equilibrium
data were also extracted from the sensorgrams to calculate the
equilibrium affinity constant using a Scatchard plot (23). A average
value of 23.6 ± 1.7 nM was returned for the
dissociation constant (Table I). The fact that this value is almost
identical to the value yielded by the kinetic analysis supports the
model used and the kinetic analysis itself.
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Table I
Summary of measured association constants for the interaction of the
coxsackievirus and adenovirus receptor D1 domain with immobilized
adenovirus type 2 fiber head
Values in parentheses are errors of the fitting procedure.
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Fig. 2A shows the binding
curves when CAR D1 was injected over a surface containing 600 RU of
immobilized fiber head. This set of data was also analyzed by numerical
integration (also called "global fitting"), which provides a
stringent test of the assumed model and returns better parameter
estimates (22). As shown in Fig. 2, the fitted curves and the
experimental data were virtually indistinguishable, using the
A + B
AB model. The returned
kinetic values were kon = 3.1 × 105 M
1 s
1 and
koff = 6.6 × 10
3
s
1, giving a Kd = koff/kon = 21 nM, in reasonable agreement with the above analysis (Table
I). Equilibrium data were extracted from the sensorgrams (Fig.
2B) and plotted according to the Scatchard (23)
representation (Fig. 2C). The straight line obtained shows that CAR D1 recognizes a single class of noninteracting binding sites,
characterized by an affinity constant of Kdeq = 26 nM, a value consistent with the result given by the
kinetic analysis of the data.

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Fig. 2.
Analysis of CAR binding to immobilized
adenovirus fiber. A, surface plasmon resonance
sensorgrams measured when CAR D1 at 212, 141, 94, 63, 42, 28, and 19 nM (from top to bottom) was injected
over a fiber head-activated surface (600 RU) at a flow rate of 50 µl/min. The response in RU was recorded as a function of time
(line) and fitted to a simple A + B
AB binding model (open squares).
B, the equilibrium level of CAR D1 bound to immobilized
fiber head (in RU) was extracted from the sensorgrams and plotted
versus the concentration (in nM) of injected CAR
D1. The data represent a complete binding isotherm. C,
Scatchard analysis. The equilibrium level of bound CAR D1
(B) was divided by the concentration of unbound analyte
(F; in nM); B/F was
plotted against B.
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The stoichiometry of the complex indicated that, on average, each
immobilized fiber head can bind a maximum of two CAR D1 molecules.
Because of the trimeric organization of the fiber head and from the
published structure of the adenovirus type 12 fiber head-CAR N-terminal
domain complex (11), a 3:1 stoichiometry would have been expected. This
discrepancy is presumably due to some of the binding sites of the fiber
head becoming inaccessible during the immobilization procedure (on
average one per fiber head trimer; see also Fig. 4).
To further check the validity of our results, we also immobilized an
adenovirus type 2 fiber protein containing the head domain plus part of
the shaft region (residues 319-582; Ref. 6) to a surface density of
700 RU. CAR D1 was injected at 20 µl/min. A dissociation constant of
30 nM and a maximum binding of 2.3 CAR D1 molecules/trimer
were observed. These values correlate very well with those found for
the fiber head alone. The similar dissociation constant indicates that
the shaft region is not involved in receptor binding, while the fact
that the average number of available binding sites is now somewhat
larger is consistent with the fact that some of these trimers will have
been attached by the shaft domain and thus have all three receptor
binding sites available.
Adenovirus Type 2 Fiber Head Binding to Immobilized CAR
D1--
In a second series of experiments, CAR D1 was immobilized on a
sensor chip and allowed to bind fiber head injected over the surface.
This system mimics the CAR presentation at a putative cell surface, and
thus may represent a more physiologically relevant assay. In
preliminary experiments we prepared different surfaces with different
amounts of immobilized CAR D1, over which fiber head was injected.
Whatever the conditions were, the sensorgrams now showed much slower
dissociation and could not be fitted to a simple A + B
AB binding model (data not shown). Visual
inspection of the sensorgrams showed that equilibrium was now much more
difficult to attain (compare the association phases of Figs.
2A and 3A), and the
formed complexes are now very stable (compare the dissociation phases
of Figs. 2A and 3A). Although the CAR D1 molecule
spontaneously dissociated from the immobilized fiber head (binding
curves returned to the base line in less than 15 min with running
buffer alone), injection of 10 mM hydrochloric acid was
required to dissociate the complex when the CAR D1 molecule was
immobilized on the surface (data not shown). To minimize all possible
artifacts, including mass transport effect and rebinding (see below),
we used a surface with a small amount of immobilized CAR D1 (200 RU)
for a more detailed analysis. Evaluation of the sensorgrams indicated
that the binding was complex and could not be fitted to a simple
A + B
AB model. Such a model
returned a
2 value (which describes the closeness of the
fit) of 23.1 (a
2 value below 10 is considered
acceptable; BIAevaluation 3.1 software handbook).

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Fig. 3.
Analysis of adenovirus fiber binding to
immobilized CAR. A, surface plasmon resonance
sensorgrams obtained when adenovirus type 2 fiber head at 50, 30, 20, 15, 10, 7, 5, 3, and 2 nM (from top to
bottom) was injected over a CAR D1-activated surface (200 RU) at a flow rate of 20 µl/min. The response in RU (full
line) was recorded as a function of time and fitted to a trivalent
binding model (open squares, see text). B,
Scatchard analysis. The levels of bound fiber head (B; in
RU) at the end of the injections of the six highest fiber head
concentrations were extracted from the sensorgrams divided by the
concentration of unbound analyte (F; in nM);
B/F was plotted against B. Although
not all points have reached equilibrium and the data do not represent a
complete binding isotherm, this analysis does give an idea about the
order of magnitude of the dissociation constant. C, CAR D1
immobilized on a sensor chip surface was saturated with fiber head
(injected at 80 nM). Subsequently, CAR D1 in solution (sCAR
at 0, 32.5, 65, 130, and 325 nM) was injected at 20 µl/min, and the response in RU was recorded. No significant increase
in RU was observed, indicating that no fiber binding sites for CAR were
available on the fiber head in the preformed complex.
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Thus, as a first approach, we measured equilibrium data. Apart from the
very highest fiber head concentrations, the measurements do not really
attain equilibrium; it appears that the CAR D1 surface is able to
capture fiber head even from dilute solutions to saturate more and more
available binding sites. Technical limitations of the apparatus used
prohibited us from using even longer contact times. However, from the
six highest fiber head concentrations we could calculate an approximate
affinity constant independently of the kinetic process (Fig.
3B). A Kd of 1.2 nM was estimated (Table II), which represents an
apparent 20-fold increase in affinity compared with the binding of CAR
D1 to immobilized fiber head. Interestingly, the stoichiometric
analysis indicated that one fiber head bound three CAR D1 molecules at
the sensor chip surface (Fig. 4), a point
consistent with the trimeric nature of the adenovirus fiber head.
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Table II
Summary of dissociation rate constants for the interaction of the
adenovirus type 2 fiber head with the coxsackievirus and adenovirus
receptor D1 domain
Values in parentheses are errors of the fitting procedure.
Kdki1 = koff1/kon1.
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Fig. 4.
Graphical representation of adenovirus fiber
binding to its receptor. The CAR D1 domain is shown in dark
gray, and adenovirus fiber head is in light gray.
A, monovalent binding reaction of the CAR N-terminal domain
to immobilized adenovirus fiber head. Shown are CAR D1 binding and
dissociating on the left and a front view of the complex on
the right. The rate constants of binding
kon and dissociation koff
and the equilibrium dissociation constant Kd of the
complex are quoted. In our experiments, about two CAR molecules could
bind simultaneously to one fiber head, indicating that on average one
of the three CAR binding sites of the fiber head was unavailable,
presumably because of the covalent cross-linking (see text).
B, trivalent binding reaction of the adenovirus fiber head
to the CAR N-terminal domain tethered to a surface (by chemical
cross-linking to a surface like in our experiments or by a membrane
anchor to a human cell membrane). Shown are the trimeric fiber head
binding to CAR D1 on the left, the trivalent complex in the
middle, and the fiber head dissociating from CAR on the
right. In our experiments, fiber head bound three CAR
molecules simultaneously.
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We then evaluated our kinetic data using a trivalent binding model:
A + B
AB, AB + B
AB2, and
AB2 + B
AB3, where A is a trivalent molecule
(fiber head in this context) that binds to B, a monomeric
component (here CAR D1). Global fitting of the data using this model
returned a
2 value of 3, indicating that the fitting
procedure has been considerably improved and now describes the kinetic
data much better (Fig. 3A). The initial binding step
(A + B
AB) was characterized by association and dissociation rate constants of 2.4 × 105 M
1 s
1 and
5.5 × 10
3 s
1, respectively.
Interestingly, these values are close to those found when monomeric CAR
D1 bound to immobilized fiber head, thus giving an affinity of 23 nM. Because these values did not depend on the molecules
which have been immobilized, they were not affected by the
immobilization process and thus are likely to be the true values.
Our data indicate that the first binding event was the binding of one
fiber head molecule to one immobilized CAR D1 molecule. However, the
complex (one fiber head bound to one CAR D1 with an affinity of 23 nM) was then further stabilized by the association of two
additional CAR D1 molecules, leading to the high overall affinity
observed. These two additional binding reactions at the chip surface
are characterized by on rates kon2 = 0.15 RU
1 s
1 and kon3 = 0.08 RU
1 s
1 and off rates
koff2 = 0.60 s
1 and
koff3 = 0.41 s
1 (Table II). The
overall off rate of the fiber head-3 CAR D1 complex was 2.2 × 10
4 s
1, and this represents a 25-fold
increase in stability versus the values measured for the
fiber head-1 CAR D1 complex. Taking into account the overall off rate
(2.2 × 10
4 s
1), the complex (fiber
head-3 CAR D1) was found to have an overall Kd of
2.2 × 10
4 s
1/2.4 × 105 M
1 s
1 = 0.9 nM (Table II). This value is lower than but similar to the
affinity we determined using quasi-equilibrium data
(Kdeq = 1.2 nM), suggesting that our
kinetic analysis is correct.
To demonstrate that the high apparent affinity is not due to rebinding
of soluble fiber head, we first injected fiber head (at 80 nM) over a surface containing 400 RU of CAR D1 to form a
stable complex and then injected soluble CAR D1 over the preformed complex. As shown in Fig. 3C, the preformed complex was
unable to bind significant amounts of additional CAR D1, demonstrating that each bound fiber head molecule already bound three CAR D1 molecules (i.e. there were no more binding sites available
in the preformed complex). Because the dissociation rate constant was
not increased by the presence of soluble CAR molecules in the running
buffer during the dissociation phase, these data also showed that a
possible rebinding effect did not contribute to the low dissociation
rate we measured.
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DISCUSSION |
Viruses initiate infection by attaching themselves to the surface
of a susceptible host cell and have evolved to use a variety of cell
surface molecules for this purpose. In this paper, we report an
avidity-based mechanism by which adenovirus fiber binds to the CAR D1
N-terminal Ig domain to form a highly stable complex, with a
stoichiometry of three CAR D1 molecules to one fiber head trimer. This
stoichiometry is consistent with the published crystal structure of the
adenovirus type 12 fiber head-CAR D1 complex (11).
We initially designed binding experiments in which CAR D1 molecules
were allowed to bind independently of each other to immobilized fiber
head. Both kinetic and equilibrium analyses indicated that CAR D1
recognizes its binding site on the fiber head with an affinity of
around 24 nM. However, the binding of soluble CAR to fiber head is only a poor mimic of the physiological interaction between a
cell surface and the virus. We thus adopted a more physiologically relevant system, in which CAR D1 was coupled to a solid phase, through
the flexible dextran matrix of a sensor chip (receptor molecules on a
cell surface are also "flexible" because of the liquidity of the
cell membrane). Not only does this system better represent CAR
expressed at the cell surface, but it also showed a 25-fold increase in
affinity by an avidity-based mechanism. This avidity mechanism allows
adenovirus to employ binding interactions with high individual off
rates in tandem to achieve an overall low off rate (for a graphical
explanation see Fig. 4) and give rise to highly stable cell surface
attachment (dissociation constant around 1 nM). In the case
of the entire adenovirus particle, this avidity could be further
enhanced by the fact that more fiber proteins interact simultaneously
each with three receptors. The mechanism we propose has been described
in qualitative terms by various authors (for a review see Ref. 24).
However, we believe we have for the first time analyzed it in kinetic
detail for one of the systems, adenovirus attachment to its receptor.
A cratic entropy contribution has been quantified for association
processes in solution (25), although the usefulness of the concept is
not universally agreed (26, 27). It would suggest an increase in
affinity of greater than the 25-fold we measured. However, we are
considering a process involving multiple interactions at a surface, in
our case a Biacore chip, but with analogies to a biological membrane.
We suggest that the decrease in degrees of liberty experienced by the
trimeric fiber head after binding to the first CAR D1 molecule may
account for the lower affinity we measure but feel that a more detailed
explanation, which should then also take into account thermodynamic
behavior (28), falls outside the scope of our current work.
The question arises of whether other trimeric receptor-ligand
interactions show the same avidity mechanism. The receptor of group B
adenoviruses and the possible second receptor of group F adenoviruses
are as yet unknown, but it is possible that they use a similar
mechanism. Trimeric cell attachment proteins of other viruses such as
reovirus sigma 1 protein and the long and short fibers of bacteriophage
T4 (1) are in our opinion likely to also employ a similar mechanism.
Many intercellular communication pathways involve trimeric
receptor-ligand interactions by members of the tumor necrosis factor (TNF) receptor family with TNF-like trimeric ligands. The interactions of the TNF receptor with TNF
(29) and TRAIL (TNF-related apoptosis inducing ligand) with DR5 (death receptor 5) (30) are surprisingly similar to that of the CAR D1 domain with adenovirus type 12 fiber head
(11), with the receptor binding in a groove between two monomers and
surface loops located toward the bottom of the trimer being
functionally most relevant (10, 11, 31, 32). It is therefore likely
that the same avidity mechanism we observe for adenovirus fiber head
binding to CAR also exists for ligands binding to the TNF receptor family.
Trimeric interactions also exist intracellularly. TNF receptor and many
of its homologues (for instance CD40) bind with their intracellular
domains to the C-terminal domains of tumor necrosis factor
receptor-associated factors (TRAFs; Ref. 33). TRAFs then mediate
signaling to various transcription factors (34). The binding of
trimeric TRAF2-C to monomeric and artificially trimerized CD40 has been
compared, and it was found that the affinity was 12-fold lower in the
case of monomeric CD40 (35).
The avidity mechanism can perhaps be extended to other multimeric cell
attachment molecules or whole viruses. The internalization of
adenovirus is mediated by the pentameric penton base (2), and by using
SPR, 4.2 integrin molecules have been measured to bind to each pentamer
with an individual affinity of 73 nM (36). In these
measurements, adenovirus was the immobilized ligand, and soluble
integrin was the analyte. Perhaps if the integrin were to be
immobilized and the penton base or the whole virus were used as
analyte, the same avidity mechanism as for the fiber head-receptor
interaction can be demonstrated. The affinity of rhinovirus (37) and
human echovirus 11 (38) for their receptors has been measured to be in
the micromolar range, again by immobilizing the virus and using soluble
receptor as analyte. We propose that the avidity mechanism will lead to
a much higher effective affinity of virus for a cell bearing multiple
copies of receptor. Indeed, a chimeric bivalent receptor binds to
rhinovirus with a 17-fold enhanced affinity (37). In the case of
influenza virus binding to polyvalent sialisides, the affinity
increases by 3 orders of magnitude (39) compared with the normal
millimolar affinity for monovalent binding (40).
A study by SPR in which saccharide ligands were immobilized on a sensor
chip surface was carried out by Mann et al. (41). They then
studied binding of concanavilin A to this surface and inhibition of
this binding by monovalent or multivalent competitor ligands. They
found a dissociation constant of around 1 µM for multivalent concanavilin binding to the surface, dissociation constants
of 92-290 µM for monovalent binding of saccharides to concanavilin A, and dissociation constants down to 2 µM
for polyvalent binding of polysaccharides to concanavilin A, a similar
avidity effect to the one we observe.
In conclusion, we have analyzed in detail an avidity mechanism that may
be widespread in receptor-ligand interactions. Experiments on other
model systems using the same strategy (immobilizing the receptors and
using soluble receptor-binding proteins or whole viruses) may yield
more examples of the avidity mechanism we describe. The next step in
this research is studying the attachment of whole viruses in kinetic
detail by surface plasmon resonance. This may be more difficult in
practice because of the size of whole viruses and their relative
instability compared with the isolated receptor-binding proteins. The
whole virus may not be able to reach all the receptor molecules
immobilized in the dextran surface, and their greater size may lead to
overloading of the plasmon resonance signal, whereas their instability
may lead to signal changes because of dissociation of the virus rather
than receptor-binding or receptor-dissociating events. Nevertheless,
the development of new SPR surface chemistries and immobilization
strategies may make these studies more feasible in the future.
Finally, adenovirus is investigated intensively as a candidate vehicle
for gene therapy (42). To target adenovirus to specific cell types,
much research is focused on abolishing CAR binding of adenovirus and
retargeting to other receptors (Ref. 43 and references therein). To
ensure that recognition of these alternative receptors is as efficient
as possible, it might be advantageous to mimic the mechanism of binding
to the natural receptor. For achieving the same stoichiometry and
avidity mechanism, alternative receptor binding sites should be
engineered into the trimeric adenovirus fiber head of adenovirus so
that each site is independently accessible by a receptor molecule.