From the
We have studied the kinetics and thermodynamics of a virus
interacting with its receptor using human rhinovirus serotype 3 (HRV3),
soluble intercellular adhesion molecule-1 (ICAM-1, CD54) containing Ig
superfamily domains 1-5 (sICAM-1), and surface plasmon resonance.
There were two classes of binding sites for sICAM-1 on HRV3, each
comprising about 50% of the total sites, with association rate
constants of 2450 ± 300 and 134 ± 11
M
Human rhinoviruses are small, non-enveloped RNA viruses of the
picornavirus family that have a capsid of icosahedral symmetry and are
300 Å in diameter
(1, 2, 3) . The outer
part of the capsid is constructed from 60 copies of viral coat proteins
VP1, VP2, and VP3; VP4 is located on the inner face of the
proteinaceous capsid. It has been proposed that the receptor binding
site is located in a depression or ``canyon'' encircling the
5-fold icosahedral vertices and that five receptor binding sites are
present around each of these vertices
(4) . Cryoelectron
microscopy, site-directed mutagenesis, and saturation binding studies
have confirmed these hypotheses
(5, 6, 19) . The
residues implicated in the interaction with the receptor are buried in
the canyon, which has been hypothesized to make the receptor binding
site inaccessible to antibodies and to protect the viral receptor
binding site from immune surveillance
(4) . It may also be
important that antibody binding sites have a bigger footprint than the
receptor binding site
(8) , and thus mutations in regions outside
but adjacent to the receptor binding site can contribute to the
immunological diversity among serotypes without affecting receptor
binding.
Intercellular adhesion molecule-1 (ICAM-1)
The equilibria, kinetics, and thermodynamics
of virus binding to receptors is interesting from both biological and
mechanistic points of view. The affinity constant is a measure of the
goodness of fit between a virus and a receptor and is important in
determining, together with the number of receptors bound per virion,
the effective or apparent affinity that governs the amount of virus
binding to cells in equilibrium. Affinity constants have previously
been measured for binding of purified influenza virus hemaglutinin to
sialic acid
(21) . The kinetics of virus:receptor interactions
are also of great importance, both because few processes reach
equilibrium in vivo, and because the kinetic constants are of
inherent interest and may lead to insights into the virus:host cell
interaction and to useful comparisons with other receptor:ligand
interactions. However, we are unaware of previous measurements of the
kinetic constants for virus:receptor interactions. Binding of
picornaviruses to soluble as well as cell surface receptors
destabilizes these viruses and promotes disruption of the protein
capsid and release of the viral RNA, which is thought to be important
in the infection
pathway
(7, 19, 20, 22, 23, 24) .
Thermodynamic measurements of virus:receptor interaction may yield
insights into the mechanism of destabilization.
To address these
questions, we have applied surface plasmon resonance, using the BIAcore
instrument
(25) , to the study of virus:receptor
interactions
(26) . This technology has allowed us to measure
binding of soluble, monomeric ICAM-1 to rhinovirus, and dissociation
from rhinovirus, in real time. The equilibrium constant derived from
the kinetic constants, and direct measurement of the equilibrium
constant by separation of the reactants by sedimentation, reveal two
classes of sites that differ in affinity and association rate constant.
There are dramatic differences in kinetic constants between binding of
soluble ICAM-1 to rhinovirus and to mAb. The effect of lowered pH and
receptor dimerization on kinetics and equilibria have been examined.
Furthermore, we have determined the kinetic constants at different
temperatures and found that heat is absorbed upon receptor binding,
which has implications for destabilization of the virion in
receptor-mediated disruption.
sICAM-1 was purified from the cell supernatant
by immunoaffinity chromatography with R6.5 mAb Sepharose as
described
(26) , with the modification that size exclusion
chromatography was with Sephadex G-200 in PBS, pH 8.0 (150 mM
NaCl, 2.7 mM KCl, 1.47 mM
KH
sICAM-1 expressed in baculovirus and mutant CHO cells has
similar molecular weight (M
IC1-5D/IgA was purified as described
previously
(20) .
Nonlinear curve fitting was carried out with the BIA
evaluation 2.0 program, using models of one site or two independent
sites to calculate association rate constants.
The portion of the
sensorgram that corresponds to the dissociation of sICAM-1 from the
rhinovirus or antibody surfaces was analyzed to obtain
k
Plots of the
rate of association (dRU/dt) of sICAM-1 with HRV3 versus the amount of sICAM-1 that had associated (RU) were biphasic
(Fig. 2A). Two classes of binding sites were
distinguished by linear transformation of the data
(Fig. 2A) and by nonlinear curve fitting (data not
shown). The latter method yielded poor fits for one binding site
(
The rates
(k
The amount of binding to the two classes of sites was determined
from the amount of bound sICAM-1 at the inflection point in the binding
curves at high (near saturation) concentrations of sICAM-1 and also
from Scatchard plots of the amount of binding to the two classes of
sites. Both methods suggested that the sites with higher and lower
k
We obtained
comparative kinetic data on three mAb that bind to ICAM-1. The mAb bind
to sites on ICAM-1 overlapping or nearby to the site that binds HRV,
since all three mAb block binding of HRV to rhinovirus
(10) , and
the epitopes have been mapped to IgSF domains 1 or 2
(16) (). The k
The enthalpy (
We have measured the kinetic constants for the interaction of
a virus with its receptor. We adapted surface plasmon resonance to the
study of virus-receptor interactions, and found it to be an accurate
method for measuring kinetic constants, with standard deviations that
were almost always less than 10% of the mean and with results that were
highly reproducible with independent viral and receptor preparations.
The utility of the method was enhanced by development of conditions
that allowed at least 12 successive cycles of sICAM-1 binding and
regeneration to be carried out with a single rhinovirus-sensor chip
preparation
(26) . No disruption of the virus was detected in the
determination of the kinetic and dissociation constants at 20 °C
and below, as monitored by changes in the base line or amount of
sICAM-1 bound.
The association rate constants for binding of sICAM-1
to HRV3 were low, 2450 and 130 M
The
k
We found two different
kinetic association constants for the sICAM-1 interaction with
rhinovirus. This contrasted with the results for association of three
mAb with sICAM-1, where a single k
There are several possible explanations for the two
classes of low and high affinity binding sites. One, there could be
inherent differences between the structure of binding sites on the
rhinovirus capsid. These two classes of binding sites appear to be on
the same virion rather than on different classes of virions, because
the rhinovirus sedimentation coefficient shifted gradually from 149 to
120 S as increasing amounts of sICAM-1 were added, and two different
populations of virions that were altered in sedimentation by different
concentrations of sICAM-1 were not seen
(22) . Binding of
``pocket factor'' to pockets underlying half of the sites
could produce the observed heterogeneity
(3) . Two, binding of
sICAM-1 could induce conformational changes in the rhinovirus capsid
that alters the structure of neighboring binding sites. Three, binding
of the first 2 or three molecules of sICAM-1 per pentamer might proceed
without hindrance, but binding of further sICAM-1 molecules might be
impeded. Electron microscopy of a two-domain fragment of ICAM-1 bound
to rhinovirus reveals no contacts between neighboring ICAM-1 molecules
(5); however, bound ICAM-1 projects outward from the virus and thus
might kinetically hinder binding, by requiring more precise orientation
for an ICAM-1 molecule to gain access to a neighboring binding site.
Our K
Multivalency results in an effective increase in affinity.
Multivalent chimeric molecules containing IgSF domains 1-2 or
1-5 of ICAM-1 fused to Fc portions of IgA1 or IgM exhibited lower
IC
Both
acidic pH and binding to receptor can cause disruption of rhinovirus
and may be important in the pathway of infection. However, the
combination of lowered pH and receptor binding are not synergistic and
not even additive in disruption assays, and less sICAM-1 binding to
HRV3 is seen at pH 6 than at pH 7
(19, 22) . The affinity
of sICAM-1 for HRV3 decreased 8-fold when pH was dropped from 8 to 6.5,
primarily as a result of a drop in k
The
thermodynamics of ICAM-1 interaction with rhinovirus may have important
implications for virus disruption. Most protein:protein interactions,
e.g. antibody binding to protein antigens, are exothermic
(negative
Four representative experiments are shown that used four
different preparations of sICAM-1 (from insect cells in experiments 1
and 2 and from Lec 3.2.8.1 CHO cells in experiments 3 and 4) and four
different preparations of HRV3. A range of concentrations of sICAM-1
from 1 to 10 µM was injected in each experiment as in Fig.
1B. k
A polyclonal rabbit anti-mouse Fc antibody (Pharmacia) was
coupled to dextran in the sensor chip. Anti-ICAM-1 monoclonals (32
µl, 20 µg/ml) and sICAM-1 (36 µl; 50, 100, 150, 200, 250,
or 300 nM) were consecutively injected through the surface
containing the rabbit antibody for 8 and 9 min, respectively, at 4
µl/min at 25 °C in PBS, pH 8.0. The rabbit anti-mouse Fc was
regenerated by injection of 8 µl of 100 mM HCl after each
cycle. k
Kinetic
association constants (k
Association
rate constants were determined from two different experiments for each
molecule using different sensor chips, as described in the legend to
Fig. 1B. Measurement units are the concentration of ICAM-1
sites, i.e. 2
sICAM-1
(1-10 or 1-12 µM) was injected at each
temperature as shown in Fig. 1B and kinetic constants
determined as described in Table I. Range
(k
s
. These rates are
low, consistent with binding to a relatively inaccessible site in the
rhinovirus canyon. By contrast, three monoclonal antibodies bound to
sICAM-1 with a single rate constant of 17,000-48,000
M
s
. The dissociation
rate constant for HRV3 was 1.7 ± 0.1
10
s
, giving calculated dissociation constants of
0.7 ± 0.1 and 12.5 ± 1.2 µM. Agreement was
good with saturation binding in solution, which showed two sites of
similar abundance with K
of 0.55 ±
0.2 and 5.7 ± 2.0 µM. A bivalent chimera of ICAM-1
with the IgA1 Fc region bound with K
= 50 and 410 nM, showing 17-fold enhanced
affinity. Lowering pH from 8.0 to 6.0 reduced affinity by approximately
50-fold, primarily by reducing the on rate. Thermodynamic measurements
showed that binding of ICAM-1 to HRV3 is endothermic, by contrast to
binding to monoclonal antibody. The heat that is absorbed of 3.5 and
6.3 kcal/mol for the two classes of ICAM-1 binding sites may contribute
to receptor-mediated disruption of virions, which has an activation
energy of about 42 kcal/mol.
(
)
is the receptor for the major group of
rhinoviruses
(9, 10, 11) . ICAM-1 is a membrane
protein with five immunoglobulin superfamily (IgSF) extracellular
domains, a hydrophobic transmembrane domain, and a short cytoplasmic
domain
(12, 13) . ICAM-1 is the counter-receptor for the
leukocyte integrins LFA-1 and Mac-1 and promotes a wide range of cell
interactions important in inflammation
(14) . ICAM-1 is also
utilized as a sequestration receptor for Plasmodium
falciparum-infected erythrocytes
(15) . The binding site on
ICAM-1 for rhinovirus is located primarily in the N-terminal or first
IgSF domain, with perhaps some contribution from the second IgSF
domain
(16, 17) . A recombinant soluble form of ICAM-1
truncated at the membrane that contains all five IgSF domains (sICAM-1)
has been shown to inhibit rhinovirus infection (18) and induce
irreversible modification of the viral capsid in
vitro(7, 19, 20) . High efficiency in
rhinovirus neutralization has been obtained with multivalent ICAM-1 Ig
chimeras
(20) .
sICAM-1
sICAM-1, the extracellular portion of
ICAM-1 containing IgSF domains 1-5, was expressed from the mutant
cDNA clone Y452E/F*
(18) using a recombinant baculovirus in SF9
cells as described previously
(22) . For expression of sICAM-1
after co-amplification with glutamine synthetase (GS)
(27) ,
Y452E/F* was introduced into the unique XhoI-NotI
site of PBJ5-GS
(28) , to produce pBJ5-GS/IC1-5D. A CHO
cell mutant (CHO Lec3.2.8.1.)
(29) with highly restricted
glycosylation was transfected with PBJ5-GS/IC1-5D using calcium
phosphate. After transfection, cells were grown in selective medium
with 10% dialyzed serum. Selective medium was essentially as
recommended
(30) and contained minimum Eagle's medium
without glutamine (Life Technologies, Inc.) supplemented with sodium
pyruvate (Life Technologies, Inc.), minimum Eagle's
medium-non-essential amino acid solution (Life Technologies, Inc.),
glucose (0.3% final concentration), glutamic acid and asparagine (60
µg/ml), nucleosides (7 µg/ml), and
L-methionine-sulfoximine (MSX). 25 µM MSX was
used in the first round of selection. Clones secreting sICAM-1 to the
extracellular medium were detected by enzyme-linked immunosorbent
assay
(31) . After the first round of selection, clones were
amplified using higher MSX concentrations (100 to 500 µM
MSX). Clone CHO-ICAM-1(5D) 37.2.3.2 was seeded in roller bottles with
expanded surface (Corning), and cells were grown to confluence using
selective media (150 ml) containing 500 µM MSX and 10%
dialyzed serum. Serum concentration was reduced to 5% when cells grew
to confluence and the roller bottle maintained at 37 °C and 2 rpm
for about 1 month. Medium was changed when the protein concentration
was about 80 µg/ml as monitored by sandwich enzyme-linked
immunosorbent assay.
PO
, 4.86 mM
Na
HPO
). Less nonspecific binding of sICAM-1 in
BIAcore was found after purification at pH 8.0 than at pH 7.4. Protein
concentration was determined from the extinction coefficient at 280 nm
of 0.8 ml/mg cm
(20) . The molecular weight for the
unglycosylated protein
(49, 600) was used to calculate
molarity.
) (
60,000) and
glycosylation content (not shown). sICAM-1 differing in glycosylation
had similar affinity for rhinovirus.
(
)
Virus Obtention
HRV3 was purified, washed free of
bovine serum albumin, and concentration determined
spectrophotometrically as described previously
(22, 26) .
Immobilization of Rhinovirus and Antibodies in
BIAcore
HRV3 was covalently immobilized to the dextran surface
of certified CM5 sensor chips via primary amino groups, using the amine
coupling kit (Pharmacia Biosensor AB
(26) ). Briefly, carboxylate
groups on the dextran were activated by injection of a mixture of
N-hydroxysuccinimide and
N-ethyl-N`-(dimethylaminopropyl)carbodiimide.
Purified virus diluted with 10 mM sodium acetate, pH 5.7, was
injected through the activated surface. Ethanolamine hydrochloride, pH
8.5, was injected after virus immobilization to block unreacted
N-hydroxysuccinimide esters. Running buffer in BIAcore used
for washing and the dissociation phase was PBS, pH 8.0, and was
identical to the buffer that sICAM-1 was obtained in after Sephadex
G-200 chromatography. After each cycle of sICAM-1 association and
dissociation, HRV3 was regenerated with 3 pulses of 2 or 3 min of 25
mM MES buffer, pH 6.0, separated by about 1 min of PBS, pH
8.0. Polyclonal rabbit anti-mouse Fc antibody (Pharmacia) was
immobilized (about 8,000 resonance units) using the amine coupling kit
as recommended by the manufacturer.
Analysis of the Binding Data in
BIAcore
Sensorgrams recorded during the interaction of sICAM-1
with immobilized virus or captured anti-ICAM-1 antibodies were analyzed
by the linear transformation method to obtain the kinetic
constants
(32) , using the equation dRU/dt =
k[sICAM-1] RU
- RU (k
[sICAM-1]
+ k
), where RU is resonance units.
Linear analysis of the binding data uses the slope
(k
) of a plot of dRU/dtversus R to determine the association rate constant
(k
). The equation
k
= k
[sICAM-1] + k
allows
determination of k
from a plot of
k
versus sICAM-1 concentration.
Several sICAM-1 concentrations were required, as described in the
figure legends.
. The slope of a plot of
ln(R1/R
) versus time
yielded k
, where R1 is the
initial RU and R
is RU with time. The
sensorgrams obtained for the highest ligand concentration were analyzed
to minimize rebinding during dissociation. When
k
was determined at different flow rates,
no change was detected.
Saturation Binding of sICAM-1 to HRV3 in
Solution
sICAM-1 was labeled with
[S]methionine and cysteine, purified, and
concentration determined by A
as
described
(22) . A mixture of labeled and unlabeled sICAM-1
(0.2-10 µM final concentration) was incubated with
HRV3 (2.1
10
virions/µl) in 75 µl for 1 h
at 20 or 15 °C, and 70 µl was subjected to sucrose gradient
sedimentation for 1 h at 40,000 rpm at 4 °C and fraction collection
and scintillation counting as described
(22) . sICAM-1 that
sedimented with the virus in the region of 120-149 S, and input
sICAM-1, were used to calculate the concentration of bound and free
sICAM-1 in the 75-µl reaction mixture. No sedimentation of sICAM-1
was observed when virus was omitted or when R6.5 mAb to ICAM-1 was
added.
Interaction of sICAM-1 with Immobilized Rhinovirus in
BIAcore
Human rhinovirus serotype 3 (HRV3) was covalently
coupled through amino groups to dextran that is immobilized on the gold
film on the surface of the BIAcore sensor chip. Changes in density of
the solution within the vicinity of the gold film (<1 wavelength)
can be measured, because this affects the refraction of polarized light
and the angle of the absorption maximum by the plasmon electrons of the
gold film
(25) . The density in the vicinity of the gold film
increases as virus is covalently immobilized, or ICAM-1 is
noncovalently bound. Reactions occur within a 0.12-nl dextran layer on
a sensor chip in a 60-nl flow chamber and are measured by the change in
RU. About 10,000 RU of virus were typically covalently immobilized,
corresponding to a concentration of 12.3 µM within the
dextran layer and a total of 1.5 fmol. Specificity was examined by
injecting ICAM-1 at 20 °C through flow chambers that contained
dextran that was either covalently coupled to HRV3 or mock-coupled in
the absence of HRV3 (Fig. 1A). sICAM-1 (6
µM) bound to immobilized HRV3 (454 RU), but little or no
binding was obtained when no virus was present (36 RU). Binding of
sICAM-1 (5 µM) to the viral surface was inhibited 85% when
the sICAM-1 was preincubated with R6.5 monoclonal antibody (6
µM) for 15 min at 37 °C (not shown). Reproducible
binding of sICAM-1 to immobilized HRV3 was obtained, and the virus
remained intact as shown by binding of the same amount of sICAM-1 and
no change in the base line after regeneration, for at least 12
successive cycles of binding and regeneration
(26) .
Regeneration, i.e. dissociation of the bound sICAM-1 between
successive cycles of injection of sICAM-1, was achieved at 20 °C
with 3 pulses of 25 mM MES buffer, pH 6.0.
Figure 1:
Sensorgrams recording interaction of
sICAM-1 with rhinovirus in BIAcore. Association during injection with
sICAM-1 and dissociation during injection of buffer lacking sICAM-1 are
marked by a gradual increase and decrease in resonance units
(RU), respectively. Changes in buffer are marked by sharp
changes in RU because of changes in buffer density. Regeneration in the
final segments of the plots with pH 6.0 MES buffer occurs after 1400 or
1200 s and is marked by a return to the pre-injection base line.
A, overlay plot of sensorgrams, normalized to the same
pre-injection RU, recorded where sICAM-1 (6 µM) was
injected at a flow rate of 2 µl/min and 20 °C through a
carboxymethylated dextran surface that had been coupled without
(-) or with (+) HRV3 (10,000 RU). This was followed by
an injection of PBS for 3.5 min. during which sICAM-1 dissociated from
the surface. The matrix was then regenerated with three pulses (3 min)
of 25 mM MES buffer, pH 6.0. B, overlay plot of
sensorgrams obtained from successive cycles of association,
dissociation, and regeneration. sICAM-1 (2, 4, 6, 8, and 10
µM) was successively injected through the sensor chip with
immobilized rhinovirus at a flow rate of 3 µl/min at 20 °C.
HRV3 was regenerated with 3 pulses (3, 2, and 2 min) of 25 mM
MES buffer, pH 6.0.
Determination of
the association kinetic constant (k )
requires measurement of association kinetics at several different
sICAM-1 concentrations, whereas dissociation kinetics can be measured
at all concentrations with typically the highest concentration yielding
the most reproducible data; sensorgrams showing association and
dissociation obtained in a typical experiment are overlaid in
Fig. 1B. The rate of ICAM-1 binding (dRU/dt)
slowed during the association phase, but binding of sICAM-1 (RU) did
not plateau, showing that equilibrium was not reached during the
injection period. The HRV3 was stable during the experiment, because a
very similar base line was recorded at the beginning and end of each
cycle. The ratio of the maximal RU of bound sICAM-1 to the RU of
covalently immobilized HRV3 was 0.075. Calculations using the
M
of virions (8.16
10
) and
sICAM-1
(60, 0) , and 60 binding sites per virion, yield
binding of sICAM-1 that is 14-18% of the theoretical maximum and
may indicate that this is the percentage of binding sites that are
accessible or remain active after coupling to dextran.
= 45) and excellent fits for two sites
(
= 1.8). Both the linear and nonlinear
methods gave similar k
values.
Figure 2:
Linear transformation of the association
of sICAM-1 with immobilized HRV3 and anti-ICAM-1 R6.5 mAb. The value of
dRU/dt every 10 s during the association phase was plotted
versus RU. Selected time intervals after the beginning of the
association with sICAM-1 are shown in parentheses. A,
sICAM-1 (8 µM) was injected through a sensor chip with
immobilized HRV3 at 3 µl/min and 20 °C as in Fig. 1B.
B, R6.5 mAb to ICAM-1 (25 µg/ml) was injected for 12 min
followed by sICAM-1 (1 µM) for 13 min in PBS at 20 °C
and 3 µl/min through a sensor chip containing immobilized rabbit
anti-mouse Fc antibody.
Interaction of sICAM-1 with immobilized mAb to ICAM-1 was measured
for comparison to results with HRV3. The plot for the association of
sICAM-1 with R6.5 mAb (and other mAb, data not shown) showed a single
straight line (Fig. 2B).
) for the first phase
(Fig. 3A) and second phase (Fig. 3B) of
association of sICAM-1 with HRV3 were plotted against sICAM-1
concentration. The slope of the line yielded k
with a correlation coefficient r > 0.99. The
intersection with the y axis is one method of determining
k
and yielded similar results for the
first and second phases of association of 1.7
10
s
and 1.8
10
s
, respectively. Plots of
k
versus sICAM-1 concentration
for association of sICAM-1 with mAb also yielded straight lines with
r = 0.99.
Figure 3:
Representative plots for determination of
the kinetic rate constants for sICAM-1 interaction with HRV3. A and B, linear transformations of the association phase of
sensorgrams as shown in Fig. 2A were used to obtain the slopes
(k) from the first (A) and second (B) part
of the association phase and k was plotted versus sICAM-1 concentration. The slope of these curves obtained by
linear regression equals k. C, a representative
linear transformation of the sensorgram from the dissociation phase
obtained with 10 µM sICAM-1. R1, resonance units
at beginning of dissociation; Rn, resonance units at the
indicated time.
Plots of dissociation of sICAM-1 from HRV3
versus time which have a slope =
k yielded a straight line
(Fig. 3C). No change in k
was observed when the injection rate during dissociation was
increased to 8 µl/min (not shown). Plots of dissociation of sICAM-1
from mAb were also linear and yielded k
that did not change with injection rate. Invariance with
injection rate for dissociation from HRV3 and mAb suggested there was
no significant rebinding of dissociated sICAM-1.
Kinetic Rate Constants
The kinetic rate constants
for sICAM-1 interaction with HRV3 were highly reproducible.
Representative data obtained with four different HRV3 preparations and
with four different preparations of sICAM-1 from CHO cells and insect
cells are shown in . The association rate constant for the
first phase of association,
k, was 2450 ± 300
M
s
. This is slow
relative to other studied association rate constants, including mAb
(see below). The rate for k
was 18-fold slower than for
k
. The
k
rate constant was 1.67
10
± 0.1
10
s
. The equilibrium dissociation constant
(K
) was calculated from
k
/k
, using
k
to obtain
K
1 and
k
to obtain
K
2. These values were 0.69 ± 0.09
µM and 12.5 ± 1.2 µM, respectively.
comprise about 40-50% and
50-60% of the total sites, respectively.
of
sICAM-1 for the three mAb was 17,000-47,800
M
s
or 7-20-fold
faster than k
for HRV3. By
contrast, the k
values for HRV3 and the
three mAb were quite similar.
Affinity in Solution
We compared our measurements
with classical techniques for measuring affinity in solution. HRV3 (1.2
nM) was incubated with varying amounts of
S-sICAM-1 for 1 h at 20 or 15 °C, chilled on ice, and
virus-bound sICAM-1 was separated from free sICAM-1 by sucrose gradient
sedimentation at 4 °C. The Scatchard plot (Fig. 4) showed two
classes of binding sites that differed in affinity.
K
1 and K
2
determined from three independent experiments at 20 °C
(±S.D.) were 0.55 ± 0.2 µM and 5.7 ±
2.0 µM, respectively.
K
and
K
at 15 °C were 0.84
µM and 6.9 µM. The high affinity sites
comprised 48% of the total at both temperatures.
Figure 4:
Equilibrium binding of sICAM-1 to HRV3 in
solution. HRV3 (2.1 10
virions/µl) was
incubated with varying concentrations of
S-sICAM-1 for 60
min at 20 °C, chilled on ice, subjected to sucrose gradient
sedimentation, and fractions were scintillation counted. Radioactivity
that co-sedimented with the virus was used to calculate bound
sICAM-1.
Effect of pH on the Interaction of sICAM-1 with
Rhinovirus
Rhinoviruses are labile to pH lower than
6.0
(33) . Lowering pH below 7.0 decreases the amount of sICAM-1
that associates with rhinovirus as shown by co-sedimentation in sucrose
gradients
(22) . We tested the effect of lowered pH on the
kinetics and equilibria of ICAM-1 binding to HRV3 (I). The
affinity of HRV3 for sICAM-1 (inverse of
K) was 1.8-fold lower at pH
7 than pH 8. A decrease from pH 7 to pH 6.5 resulted in a further
4.6-fold drop in affinity, primarily due to a 3.2-fold drop in
k
. At pH 6 there was a
substantial further drop in affinity, as shown by less binding of
sICAM-1 at the end of the association phase with 10 µM
sICAM-1 than at higher pH (I). The
k
was 2-fold faster at pH 6.0 than at pH
6.5, and too little sICAM-1 was bound for measurement of
k
. The affinity roughly dropped 6-fold
from pH 6.5 to 6.0, since the same amount of sICAM-1 was bound at the
end of the association phase at pH 6.5 with 5 µM sICAM-1
(52 RU) as at pH 6.0 with 30 µM sICAM-1 (48 RU). Thus,
from pH 8 to pH 6 there was a marked drop in affinity of approximately
50-fold. There was no disruption of HRV3 at pH 6 to 8 at 20 °C,
since there was no change in the base line of immobilized rhinovirus,
and since binding of sICAM-1 was reproducible in repeat experiments
with the same sensor chip, and since full sICAM-1 binding activity was
obtained in binding experiments at pH 8 that followed those at pH 6
(data not shown). Indeed, it should be noted that the ICAM-1 binding
sites on HRV3 were routinely regenerated at the end of every
association and dissociation cycle with pulses of pH 6 buffer.
Effect of ICAM-1 Dimerization on the Interaction with
Rhinovirus
A dimeric ICAM-1 chimera, with ICAM-1 IgSF domains
1-5 fused to the hinge, CH2, and CH3 domains of the IgAI Fc
region, has increased efficacy in neutralizing and disrupting
rhinovirus
(20) ; however, its affinity has not been measured
previously. We measured the kinetics of the interaction of this
IC1-5D/IgA chimera with HRV3 (). The kinetics were
measured in terms of the concentration of the ICAM-1 moiety, i.e. for the dimeric chimera 2 the molar concentration. The
k
k
kinetic constants
were 1.6- and 2.8-fold faster for IC1-5D/IgA than for sICAM-1,
respectively, whereas k
was 7.6-fold
slower. This resulted in a 17-fold higher affinity for IC1-5D/IgA
than for sICAM-1.
Thermodynamics of the Interaction of sICAM-1 with HRV3
and R6.5 mAb
Thermodynamics of the interactions were determined
by measuring kinetic constants at temperatures ranging from 10 to 25
°C ( and Fig. 5). For the interaction of sICAM-1
with both HRV3 and R6.5 mAb, k and
k
increased with increasing temperature.
For HRV3, k
and
k
increased more than
k
whereas for R6.5 mAb
k
increased less than
k
. The data fit straight lines on
Arrhenius plots (Fig. 5) except for the k
for HRV3 at 25 °C. This value was disregarded in the plot
because of decrease in the HRV3 RU base line at the end of each cycle
at 25 °C, suggesting virus disruption; at 30 °C substantial
disruption of HRV3 occurred in the presence of sICAM-1 (26). The
K
and
K
for HRV3 decreased by 1.2-
and 1.4-fold as temperature increased from 10 to 20 °C. By
contrast, the K
for R6.5 mAb increased
from 10 to 20 °C by 1.3-fold.
Figure 5:
Activation energy for k and
k. Kinetic constants were determined in the BIAcore machine
equilibrated to 10, 15, 20, or 25 °C. A, for HRV3
association, kinetic constants were determined using ICAM-1
concentrations of 1-12 µM and dissociation constants
were determined using the two highest concentrations. Plots obtained
with the kinetic constants determined for the first (open
squares) and second phase (open triangles) of the
association of sICAM-1 with HRV3 are shown. The mean and range are
shown for two independent experiments. B, for R6.5 mAb,
constants were determined as described in Table II in three different
experiments where sICAM-1 was injected at 1, 2, or 4 µl/min through
sensor chips with about 900 RU of R.6.5 mAb captured with anti-mouse
Fc. Nine concentrations of sICAM-1 between 100 and 500 nM were
used in each experiment. k was determined from the sensorgrams
obtained when sICAM-1 was injected at 350, 400, 450, and 500
nM at flow rate of 4 µl/min. Mean and S.D. are shown for
three experiments (k) and four dissociations (k).
Activation energy is obtained from the slope of the plots and T is in Kelvin.
The activation energy
(E) for association and dissociation was
determined from Arrhenius plots (Fig. 5). The highest
E
were for k
for sICAM-1 binding to HRV3 and k
for dissociation of sICAM-1 from R6.5 mAb. The E
for dissociation of sICAM-1 was markedly higher with R6.5 mAb
than with HRV3.
H°) and free energy
(
G°) for the association of sICAM-1 with HRV3 and
R6.5 at 20 °C were determined from the activation energies and
affinity constants, respectively (). The HRV3-sICAM-1
interaction is an endothermic process, as shown by the positive
H° for the reaction. Association of sICAM-1 with the
second class of binding sites on HRV3 was markedly more endothermic
than with the first class. Association of sICAM-1 with R6.5 mAb was
exothermic, with a negative
H°. The entropy term
(T
S°) was positive for both interactions,
but was higher for the interaction of sICAM-1 with HRV3 than with R6.5.
s
. Since we are unaware of any previous
measurements of the kinetic constants for virus binding to receptors,
it is useful to compare kinetic measurements for other protein-protein
interactions. Association rate constants for antibody binding to
proteins in solution or on the cell surface range from 10
to 2
10
M
s
(34), consistent with our range of 2-5
10
M
s
for three ICAM-1 mAbs. On rates for the IgE receptor and insulin
receptor are 6
10
and 3
10
M
s
(34) ,
respectively, whereas the adhesion molecules CD2 and CD48 interact with
an on rate > 10
M
s
(35) . The latter interaction has an
affinity similar to that of ICAM-1 for HRV3. There are two interesting
possible explanations for why association of sICAM-1 with rhinovirus
proceeds 1-2 orders of magnitude more slowly than typically found
for protein:protein interactions. The location of the rhinovirus
binding site in a depression in the viral capsid may make it relatively
inaccessible and require precise orientation of the ICAM-1 for binding.
Alternatively, there may be a requirement for a conformational change
in the binding site on rhinovirus for ICAM-1 to bind.
of 1.67
10
s
found for sICAM-1 and HRV3 translates to a
t or lifetime of the receptor-ligand bond of 6.9 min. The
k
was not unusual and was similar to that
found for three different mAb to ICAM-1.
was
found. Of the binding sites on HRV3, 40-50% bound with the faster
on rate and the remainder with the slower on rate. Using our kinetic
constants, we calculated the equilibrium dissociation constant of
sICAM-1 for HRV3. The K
is 0.69 ±
0.09 µM and 12.5 ± 1.2 µM for the two
classes of binding sites. Equilibrium binding measurements in solution
yielded a biphasic Scatchard plot. The K
of 0.55 ± 0.2 µM and 5.7 ± 2.0
µM were in good agreement with the BIAcore results. There
were almost equal numbers of low and high affinity sites, as also found
with BIAcore.
measurements may be compared
with previous determinations of the IC
for inhibition by
sICAM-1 of virus binding to cells, infection at high multiplicity of
infection or plaque-forming units
(7, 18, 20) .
For HRV3, the IC
for inhibition of binding to purified
ICAM-1 on a substrate was 3.5 µM, and the IC
for infectivity at high MOI and in plaque-forming assays was 1.2
µM and 0.4-0.3 µM,
respectively
(7, 20) . The high affinity
K
of 0.7 µM is close to
these IC
values and suggests that occupancy of these sites
may be sufficient for inhibition of these processes. Inhibition of
binding to cells is obtained at lower IC
values of 45
nM(20) , suggesting that occupancy of only a few sites
is sufficient to inhibit virus attachment. IC
values for
HRV54
(18) and HRV14
(20) are lower than for HRV3, and
these viruses may have correspondingly lower K
for
sICAM-1.
than sICAM-1 in assays of binding to cells, disruption,
and plaque-forming units
(20) . We measured the kinetics and
K
for the IC1-5D/IgA chimera in
units of concentration of the ICAM-1 moiety, for fair comparison with
sICAM-1. The chimera, like sICAM-1, exhibited two
k
. The
k
and
k
were 1.6- and 2.8-fold
faster than for sICAM-1, respectively. Theoretically,
k
for sICAM-1 and IC1-5D/IgA are
predicted to be the same
(34) . Bivalent binding is predicted to
result in a decreased k
, in agreement
with the finding that k
was 7.5-fold
lower for IC1-5D/IgA than for sICAM-1. The
K
for IC1-5D/IgA were 50 and 411
nM, 17-fold lower than for sICAM-1. This compares with
IC
values for IC1-5D/IgA of 7.5-, 12.6-, or 187-fold
lower than for sICAM-1 for virus binding, disruption, or plaque-forming
units, respectively
(20) . The IC
values for these
three assays were 1.6-38 nM. Compared with the high
affinity K
of 50 nM or average
K
of 230 nM, this suggests that
50% inhibition occurs when substantially less than 50% of the
sites/virion are occupied. The K
for
binding of HRV14 and HRV15 to HeLa cells is in the range of
10
M(6) . Comparisons with our
values of about 10
M and 10
M for monovalent and bivalent receptor binding clearly
suggest that binding of HRV to cells is highly multivalent.
.
This is a very dramatic change in affinity over a small change around
neutral pH and may suggest either a charged residue such as histidine
that is important in binding or a cooperative change in capsid
conformation that affects the receptor binding site.
H) and thus have higher affinity as the
temperature is decreased
(34) . The interaction of R6.5 mAb with
sICAM-1 was an example of this. By contrast, interaction of HRV3 with
ICAM-1 was endothermic; i.e. heat was absorbed by the
sICAM-1-HRV3 complex. Both interaction with R6.5 mAb and HRV3 resulted
in an increase in entropy, consistent with a hydrophobic interaction
that results in increased disorder of water molecules. We have
previously measured the activation energy for disruption of rhinovirus
as
42 kcal/mol
(22) . Binding of sICAM-1 to rhinovirus
accelerates the rate of disruption, and thus partially destabilizes the
virion. Part of the heat absorbed when sICAM-1 is bound may contribute
to lowering the activation barrier for disruption. The enthalpy for
binding to the sites with slower k
of 6.3
kcal/mol is substantially higher than for binding to sites with faster
k
of 3.5 kcal/mol. Binding to the former
class of sites would be predicted to make a greater contribution to
disruption because of the greater enthalpy. Since there are a total of
60 sites/virion, occupation of only a fraction of them would result in
an increase in enthalpy similar to that of the activation energy of 42
kcal/mol. It may be significant that binding to the sites with lower
enthalpy would precede kinetically binding to the sites with higher
enthalpy; this may be important in the pathway of virus disruption and
infection. Studies of virus disruption in BIAcore may lead to further
insights into the physicochemistry of this process.
Table:
Kinetic and dissociation constants for sICAM-1
and HRV3
and
k
were obtained from the
analysis of the first and second part of the biphasic association
phase, respectively. Dissociation constants are the average of
measurements from the dissociation phase of the two highest sICAM-1
concentrations. K
were calculated from
the kinetic constants; K
=
k
/k
and K
=
K
/k
.
Standard deviations are in parentheses.
Table:
Kinetic and dissociation constants for sICAM-1
and mAb
is from a single or two
experiments, with range in parenthesis. k
is the average and s.d. of measurements from the dissociation
phase from three cycles where 300, 500, and 750 nM sICAM-1
were injected at 4, 8, and 30 µl/min, respectively.
Table:
Effect of pH
on kinetic and dissociation constants for sICAM-1 and HRV3
) are averages of
two experiments with range in parentheses; k
constants are average of four measurements with S.D. in
parentheses. The buffer for injection of ICAM-1 and the dissociation
phase was PBS adjusted to the indicated pH. sICAM-1 was at a range of
concentrations from 1 to 10 µM, pH 8.0; 1 to 15
µM, pH 7.0; 5 to 30 µM, pH 6.5; and 10 to 30
µM, pH 6.0. k
,
k
, and
k
were determined and
K
calculated as described in Table I. The
last column shows the amount of sICAM-1 that was bound in RU after 13
min of injection at 10 µM. The amount of HRV3 immobilized
was 11,000 RU.
Table:
Kinetic and
dissociation constants for sICAM-1 and IC1-5D/IgA
molar concentration for
IC1-5D/IgA. Concentrations injected ranged from 1 to 8 µM for sICAM-1 and 0.5 to 4 µM for IC1-5D/IgA. The
experiments were done in parallel at 4 µl/min using PBS, pH 8.0.
Dissociation constants were determined from the two highest protein
concentrations in each experiment, and one additional experiment with
the highest protein concentration at 10 µl/min. The standard
deviation is in parentheses.
Table:
Kinetic and association constants for
HRV3-sICAM-1 interaction at four different temperatures
) or standard deviation
(k
) are shown in parentheses.
Table:
Thermodynamic parameters
H° was obtained from the difference between the
activation energy for the association and dissociation reaction, which
is equivalent to the activation energy for the equilibrium association
constant.
G° was determined from the affinity
constants at 20 °C (
G° = -RT ln
K
), and T
S° was
calculated from the difference between
H° and
G°. Data from the first (1) and second (2) phase of
the association of sICAM-1 with HRV3 are shown.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.