From the
Knowledge of the kinetic binding characteristics is often
critical to the development of ligand/receptor structure-activity
relationships. To better understand the contribution of each of the
subunits to ligand binding in the multimeric interleukin-2 receptor
system, we have previously prepared stable solution complexes of the
The specificity and stoichiometry of receptor subunit
association are the critical parameters that determine both the ligand
binding and signaling properties for the hematopoietin receptors. In
systems where both homomeric and heteromeric subunit association may
occur via ligand-dependent or independent mechanisms and where multiple
ligands may share a single common receptor subunit, understanding the
nature of receptor aggregation is crucial to developing ligand/receptor
structure-activity relationships. As a general approach to the stable
solution assembly of cytokine receptors, we have employed coiled-coil
molecular recognition to generate both homomeric (1, 2) and heteromeric (2) interleukin-2 receptor
complexes (IL-2R
SPR
is emerging as a sensitive and rapid method for the real time analysis
of macromolecular interactions (6, 7) and has been
successfully employed for quantitation of receptor-ligand interactions
including human growth hormone and interleukin-5(8, 9) ,
members of the helical cytokine/hematopoietin receptor family. Although
this method is very convenient for acquiring kinetic data, it can also
be subject to a variety of experimental artifacts that may confound the
analysis of data and interpretation of results(10) . In this
study we have attempted to critically evaluate this technique for its
ability to provide both kinetic and equilibrium constants for soluble
IL-2R
Purified
IL-2R
Sensorgrams were analyzed by
nonlinear least squares curve fitting using BIAevaluation 2.0 software
(Pharmacia). A single-site binding model (A + B = AB) was used for analysis of interaction of IL-2
analogs to IL-2R
To analyze the association
phase, the equation R
The
two-site dissociation model, AiBj = Ai + Bj, was employed for analysis of sensorgrams on IL-2R
Since binding of the IL-2 analogs
reached a constant equilibrium value during the injection on all three
receptor complex surfaces, this equilibrium value (R
In previous studies of coiled-coil IL-2R complexes,
competitive radioligand binding assays indicated that these complexes
bound IL-2 with affinities that were characteristic of cell surface
receptors(1, 2) . In particular, the heteromeric
Therefore, we prepared biosensor
surfaces for each of the IL-2R
In contrast, another IL-2 analog possessing a
The dissociation constant
determined from the ratio of the kinetic constants for the
SPR analysis of the T41P analog provided similar
results (Fig. 4C). The kinetic constants () and the dissociation constants () confirm that this analog has a much higher
affinity to the
By employing coiled-coil molecular recognition, we have succeeded in
preparing stable homomeric and heteromeric solution complexes of IL-2R
ectodomains. In this study, we have used SPR methodology (BIAcore) to
examine the binding parameters of IL-2 and IL-2 analogs to
The
kinetic constants determined for IL-2 to all three surfaces were
generally in accordance with literature values (where available)
obtained in kinetic binding experiments of IL-2 to cell surface
receptors. In particular, the association and dissociation rate
constants measured for the high affinity
When IL-2
analogs possessing receptor subunit specific mutations were examined,
binding to surfaces differed from IL-2 according to the nature of the
mutation. Primarily as a result of an increased off-rate, the analog
with an
The influence of the lysine exchange at position 20 of IL-2
on
Taken together, these results not
only indicate that the IL-2R
Kinetic rate constants
determined as described under ``Materials and Methods'' from
sensorgrams as depicted in Figs. 2-4. NA refers to values that
could not be accurately determined because they exceeded the
limitations of the instrumentation. ND refers to no specific binding
detected. T41P and D20K are IL-2 analogs with
Dissociation constants as determined from the ratio of the kinetic
rate constants (K
We thank Russ Granzow and Chris Whalen (Pharmacia
Biosensor) for advice and discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
- and
-subunits. In this study, we have employed surface
plasmon resonance biosensor methodology (BIAcore
) to
evaluate both the kinetic and equilibrium binding constants for these
complexes. The structural nature of the complexes facilitated
immobilization on the sensor surfaces in a manner that minimized
interference with ligand interactions. The interleukin-2 receptor
complex surfaces displayed excellent binding capacity and stability
toward regeneration. In all cases where the binding constants were
measurable, the values determined for interleukin-2 were in good
agreement with those previously determined by other methods. When
interleukin-2 analogs with receptor subunit specific mutations were
employed, the binding parameters were consistent with the nature of the
mutations. The combination of coiled-coil-mediated solution assembly
and surface plasmon resonance analysis of ligand binding provides a
powerful approach to the study of multimeric cytokine receptor systems.
(
)
cc). These complexes
bound IL-2 with characteristic cell surface affinities in competitive
radioligand binding assays. Solution binding assays, however, do not
reveal the kinetic characteristics of ligand binding. In earlier
studies, the determination of the kinetic constants for IL-2 binding to
cell surface IL-2 receptors proved to be of key importance in
understanding the nature of the various physiological forms of the
receptor(3, 4, 5) . In order to determine the
kinetic ligand binding properties of the soluble IL-2R
cc
complexes and verify their equilibrium dissociation constants, we have
employed surface plasmon resonance (SPR) biosensor technology.
cc complexes in a system where comparable information exists
for cell surface receptor complexes. In addition, we have determined
similar values for IL-2 analogs that contain receptor subunit specific
mutations. The results of these analyses support both the utility of
SPR and the feasibility of coiled-coil solution assembly of receptor
complexes.
Protein Expression and Purification
The IL-2
receptor complexes were expressed in insect cells and purified as
described previously(1, 2) . Recombinant IL-2 and IL-2
analogs were expressed in Escherichia coli, refolded, and
purified as described previously(4, 11, 12) .
Protein concentrations were calculated from A values determined in 6 M guanidine HCl(13) .
SPR Reagents and Biosensor Surface Preparation
SPR
instrumentation (BIAcore), CM5 sensor chips and amine coupling
reagents containing N-hydroxysuccinimide, N-ethyl-N`-(3-diethylaminopropyl)carbodiimide, and
ethanolamine HCl were obtained from Pharmacia Biotech Inc.
cc complexes were diluted to a concentration of
30
nM in NaOAc buffer (10 mM, pH 4.5) and coupled to the
dextran-modified gold surface of a CM5 sensor chip using the
manufacturer's amine-coupling chemistry as described in the
BIAcore systems manual. Briefly, the dextran surface of sensor chip was
first activated with N-hydroxysuccinimide/N-ethyl-N`-(3-diethylaminopropyl)carbodiimide
(15 µl) followed by the addition of receptor complexes (15-45
µl). The remaining activated groups were blocked by injection of
ethanolamine (35 µl). Employing these conditions, surfaces
containing densities of 500-2000 resonance units (RU) of IL-2R
cc
complexes were generated. Prior to data collection, several methods for
surface regeneration after ligand binding were evaluated. It was found
that injection of 10 mM HCl (4 µl) could efficiently
remove the bound proteins and preserve the binding capacity of sensor
chip surface. The IL-2R
cc surfaces were stable to more than a
hundred binding and regeneration cycles.
Collection and Analysis of Sensorgrams
Prior to
SPR analysis, IL-2 and analogs were dialyzed against phosphate buffer
(10 mM sodium phosphate, pH 7.4, 150 mM NaCl), and
the protein concentrations were determined and then diluted to the
desired concentrations in HBS buffer (10 mM HEPES, pH 7.4, 150
mM NaCl, 3.4 mM EDTA, 0.005% surfactant P-20)
containing 100 µg/ml bovine serum albumin. Five to eight serial
dilutions of each protein were injected over the IL-2Rcc surfaces
at a flow rate of 8 µl/min. Sensorgrams were recorded and
normalized to a base line of 0 RU. Equivalent volumes of each protein
dilution were also injected over a mock, nonprotein, blocked surface to
serve as blank sensorgrams for subtraction of bulk refractive index
background. Each determination was repeated 3-5 times. For the
analysis of association rate constants, biosensor surfaces of less than
1000 RU of IL-2R
cc complexes were employed. For the determination
of dissociation rate constants, surfaces of higher receptor densities
were sometimes employed, and HBS buffer containing 1 µM
cc or
cc complex was injected during
the dissociation phase to capture released ligand and minimize
rebinding to the biosensor surface.
cc and
cc surfaces. The equation R
= R
exp(-k
(t - t
)) was used for dissociation phase, where R
was the amount of ligand remaining
bound (in RU) at time t and t
was the
beginning of dissociation phase. The final dissociation rate constant, k
was calculated from the mean of those values
of each injection in an injection series.
= R
(1 - exp(-k
(t - t
))) was employed where R
was the amount of ligand bound in RU at
equilibrium, t
was the time that injection
started, and k
= k
C
+ k
, where C was the concentration of
protein ligand injected over the sensor chip surface. The association
rate constant, k
, was determined from the slope
of a plot of k
versus C.
cc surfaces. The following equation was used: R
= R
exp(-k
(t - t
)) + (R
- R
) exp(-k
(t - t
)), where binding to receptor site 1
(dissociation rate constant, k
) in RU is R
and to site 2 (k
) is R
- R
. For the
association phase analysis on the
cc surface, the data
were fitted to both a two-site and a single-site model. When low ligand
concentrations were used and when IL-2 analogs with receptor
subunit-directed mutations were tested, results fitted to the
single-site model proved superior.
) was employed for Scatchard analysis (14) of the binding data. After subtraction of the background RU
due to bulk refractive index changes (determined for each dilution by
injection over a blank, blocked surface), R
/C was plotted versus R
. The dissociation
constants and surface site densities were estimated by nonlinear least
squares curve fitting using the program EBDA/RADLIG (version 4,
BIOSOFT) (15) employing the conversion factor 1000 RU = 1
ng IL-2/mm
(BIAcore systems manual).
cc complex contained a pseudo high affinity site having
an equilibrium dissociation constant of about 300 pM. To
further investigate the binding characteristics of these receptor
complexes, we have employed SPR biosensor (BIAcore) analysis as a
method for the determination of both the kinetic and equilibrium
binding constants. In this system, the binding of a ligand to a
receptor immobilized on a dextran/gold surface can be visualized in
real time by surface plasmon resonance detection, and the resulting
binding curves can be used to derive the kinetic parameters of the
interaction(6, 7) .
cc complexes (
cc,
cc,
cc) and studied the binding of IL-2 as
well as IL-2 analogs containing specific receptor subunit-directed
mutations in order to evaluate this method and compare the results to
similar findings determined by other techniques.
Surface Preparation and Stability
Using the standard
amine coupling chemistry, the purified IL-2Rcc complexes were
efficiently immobilized onto the sensor's activated dextran
surface. After preparation, each surface was tested for its capacity to
bind IL-2. All of the IL-2R
cc surfaces displayed a high ligand
binding capacity relative to total surface RU density as well as long
term stability. Typically, more than 80% of the original binding
capacity was preserved after 100 cycles of ligand binding and
regeneration. In contrast, surfaces prepared with the individual,
uncomplexed
- and
-IL-2R subunit ectodomains, using the same
amine coupling methods, were found to have reduced binding capacity and
stability.
(
)This is likely due to the
nonspecific nature of the amine coupling technique and associated
perturbation of the ligand binding site. Since the coiled-coil
complexes contain an extended lysine-rich stalk (
40 lysine
residues on the surface of the 75-Å triple helix), covalent
attachment through the coiled-coil domain is highly probable and would
result in the receptor ectodomains being oriented away from the linkage
site (Fig. 1). Such immobilization would offer little
interference to ligand binding and provided a clear advantage over the
random coupling of the individual receptor ectodomains.
Figure 1:
Biosensor immobilized IL-2Rcc
complexes. An illustration of IL-R
cc complexes immobilized by
covalent attachment through the coiled-coil domain to the
functionalized gold/dextran surface. Shown are the heteromeric
cc complexes containing two
-subunit ectodomains (darkgray) and one
-subunit ectodomain (lightgray) on the biosensor surface. Blackshapes represent IL-2. The illustration is not drawn to
scale.
SPR Analysis of Ligand Binding to IL-2R
Sensorgrams were obtained at a variety of concentrations
of IL-2 over different IL-2Rcc
Surface
cc surface densities for each
complex. For the
cc surface, representative sensorgrams
(100-500 nM IL-2) are shown in Fig. 2A. The association phase (340-540 s)
was analyzed by nonlinear least squares curve fitting as described
under ``Materials and Methods'' to yield k
values at each concentration. A plot of k
versus concentration of IL-2
provided a straight line (Fig. 2D) with a slope
equal to the association rate constant (k
). The
value of k
for IL-2 binding to the
cc
surface () was determined to be 1.06 ± 0.03
10
(M
s)
. The
dissociation phase (580-700 s) was also analyzed by the nonlinear
least squares curve fitting. The dissociation rate constant, k
, was calculated from the fitting of the first
30 s of true dissociation. The k
for IL-2 from
cc surface was calculated as 3.47 ± 0.33
10
s
(). The
apparent equilibrium dissociation constant (K
) determined from the
ratio of these two kinetic constants (k
/k
was 32.7
nM.
Figure 2:
Analysis of ligand binding to the
cc surface. Sensorgrams (relative response in RU after
background subtraction versus time in sec.) of IL-2 (A, lower to uppercurve: 100, 200,
300, 400, and 500 nM); D20K (B, lower to uppercurve: 50, 100, 200, and 300 nM); and
T41P (C, lower to uppercurve: 100,
200, 300, 400, and 600 nM) injected over an
cc
surface at a flow rate of 8 µl/min. PanelD,
plots of k (or k
C + k
) versus concentration (C)
for IL-2 (
), T41P (
), and D20K
(
).
This value compared favorably to the K (30 nM)
obtained from plotting the R
obtained for each
ligand concentration in a Scatchard analysis (see Fig. 5A). These two dissociation constants obtained from
the SPR data () were also similar to that obtained
in previous competitive radioligand assay (45
nM/
-subunit) for the same complex(2) .
Figure 5:
Scatchard plots of surface bound ligand.
Plots of R/concentration (RU of ligand bound at
equilibrium after background subtraction/concentration of ligand
injected) versus concentration for IL-2 on the
cc
surface (A); IL-2 on the
cc surface (B);
IL-2 on the
cc surface (C); and T41P on the
cc surface (D). Equilibrium dissociation
constants and relative binding sites determined from this data using
RADLIG/EBDA as described under ``Materials and Methods.'' The
results (see Table II) obtained using this analysis were as follows:
for IL-2 (panelC) K
= 0.56 ± 0.14 nM, K
= 29.6 ± 8.8 nM, B
/B
= 1.17
± 6%; for T41P (panelD) K
= 11.0 ± 1.9 nM, K
= 787 ± 282 nM, B
/B
= 0.68
± 17%.
To check
the specificity of the cc surface, we determined the binding
characteristics of an IL-2 analog containing a point mutation (Pro for
Thr-41, T41P) that primarily inhibits
-subunit binding (12). When
the T41P analog was injected over
cc surface (Fig. 2C), we observed a large decrease in
affinity. Kinetic analysis of T41P binding to IL-2R
cc
surface indicated that this mutation has little influence on the rate
of association (). For analysis of the dissociation
phase, 1 µM
cc complex was included in the
buffer to capture released ligand since binding of T41P to the
-subunit alone was very weak. Nevertheless the proline for
threonine 41 exchange increased the dissociation rate constant to the
extent that it could not be accurately determined by this technique (>0.1 s
, ). The
equilibrium dissociation constant determined from Scatchard analysis of
the sensorgrams (K
= 650 nM, ), however, was
similar to that previously determined (K
= 880 nM) in solution competitive binding
assays(12) .
-subunit specific mutation (Lys for Asp-20, D20K) (16, 17) displayed little difference in binding
parameters to this surface when compared with IL-2 (Fig. 2B, Tables I and II). Therefore, SPR
results on the
cc surface were consistent with previous
findings for both the cell surface
-subunit and the soluble
cc complex. It should be noted that k
for IL-2 binding to the
cc surface was approximately
10-fold slower than we previously reported for k
to the cell surface
-subunit (1.2
10
(M
s)
)(4) . This
difference could reflect real differences inherent to the SPR biosensor
method or simply error in the determinations, since both association
rate constants are very rapid and approach the limitations of the
respective experimental procedures.
SPR Analysis of Ligand Binding to IL-2R
We next examined the ligand binding properties to an
IL-2R cc
Surface
cc (1) surface prepared in similar fashion (Fig. 3A). Due to the lower affinity of this
subunit, we examined the association phase over a concentration of
0.12-2 µM. The k
for IL-2 () was approximately 5-fold slower than that to the
-surface, while k
was too rapid to be
determined. Scatchard analysis of the R
values at
each concentration (see Fig. 5B) provided a K
value of 407
nM. This value compares favorably with the previously
determined solution K
value of 300
nM(1) . These dissociation constants would imply an
off-rate constant of
10
s
, a
value that exceeds the detection limit of the instrumentation.
Figure 3:
Analysis of ligand binding to the
cc surface. Sensorgrams (relative response in RU after
background subtraction versus time in sec.) of IL-2 (A, lower to uppercurve: 250, 500,
750, 1000, and 1500 nM); D20K (B, 1, 2, 3, and 4
µM), and T41P (C, lower to uppercurve: 100, 250, 500, 750, and 1000 nM) injected
over an
cc surface at a flow rate of 8 µl/min. PanelD, plots of k (or k
C + k
) versus concentration (C) for IL-2 (
), T41P (
).
When
the IL-2 analog D20K was examined, no binding was detected to the
cc surface using concentrations up to 4 µM of
ligand (Fig. 3B). This is consistent with the
previous reports that this mutation is
-receptor subunit-specific.
In contrast, both the on-rate constant () and the K
()
for the T41P analog binding to the
cc surface (Fig. 3C) were much closer to the values found
for wild-type IL-2. Again, this is in accordance with the nature of
this mutation being primarily
-receptor subunit-directed with
little influence on the binding to the
-subunit. Therefore, as was
the case with the
cc surface, the ligand-binding parameters
determined by SPR for the
cc surface were in agreement with
previous reports of cell surface and solution binding for IL-2 and the
analogs examined.
SPR Analysis of Ligand Binding to IL-2R
Since our interest is the solution
assembly of heteromeric IL-2 receptor complexes, it was our goal to
determine if SPR would be useful in the characterization of the kinetic
and equilibrium binding properties of complexes containing more than
one IL-2R subunit. We have prepared and characterized a heteromeric
IL-2Rcc Surface
cc complex containing two
and one
IL-2R
ectodomains (2). This complex was capable of binding IL-2 in solution
with a K
of 320 pM, a value much
higher than the K
values for either of
the individual subunits and in the range reported for the
pseudo high affinity cell surface complex(5, 18) . To
determine the kinetics of binding of IL-2 to this heterocomplex, we
prepared
cc surfaces on a CM5 chip. Fig. 4A depicts typical sensorgrams obtained for
the binding of IL-2 to an
cc surface. Since this
surface contains more than a single class of binding sites, we prepared
a low density surface (800 RU) to analyze the high affinity site while
minimizing any mass diffusion effects during the association phase.
When concentrations of IL-2 from 2-20 nM were run over
the surface and the association phases of the curves were analyzed, the
data fit a single-site binding model with a k
= 3.90 ± 0.04
10
(M
s)
better than a two-site
model. This value is
4-fold faster than that determined for the
cc and supports our previous kinetic analysis suggesting
that the
pseudo high affinity IL-2R exists preformed on the
cell surface and captures ligand in a cooperative fashion(4) .
It is likely that with the lower IL-2 concentrations employed in the
on-rate analysis, association occurs primarily to the
single
binding site in the complex. Therefore, the one-site binding model
provided superior fitting to the data. Since the on-rate constants to
both the
cc and
cc surfaces are within a
factor of five, the result obtained from the single-site analysis may
be an underestimate of the true k
to the
site in the complex. The on-rate to this site is at least
the value reported and possibly slightly faster.
Figure 4:
Analysis of ligand binding to the
cc surface. Sensorgrams (relative response in RU versus time in s) of IL-2 (A, lowerto uppercurve: 20, 30, 40, 50, and 60
nM); D20K (B, lower to uppercurve: 50, 100, 300, 400, and 500 nM), and T41P (C, lower to uppercurve: 20, 30,
40, 50, and 60 nM) injected over an
cc surface
at a flow rate of 8 µl/min. PanelD, plots of k (or k
C + k
) versus concentration (C)
for IL-2 (
), T41P (
), and D20K
(
).
For analysis of the
dissociation phase, a higher density surface (2200 RU) was prepared.
Unlike the association phase, the dissociation phase data fit a
two-site binding model better than a single site. Two k values were determined from this data, a
slower rate constant of 2.02 ± 0.07
10
s
and a faster value of 4.72 ± 0.40
10
s
(). The slower off rate corresponds to the
higher affinity
site and is considerably slower than the
values obtained from the homomeric complex surfaces, while the faster k
is similar to that previously obtained with
the
cc surface and is consistent with dissociation from the
single
site in the complex.
site was K
= 0.52
nM, a value close to the K
previously determined in solution(2) . Scatchard
analysis of the R
values at each concentration
confirmed that two sites were present in the complex (Fig. 5). The K
values obtained from this curvilinear plot were 0.56 and 30
nM (). Both of these dissociation
constants agree with the K
and K
values
previously obtained for the
and
sites, respectively (). Furthermore, the stoichiometry revealed in the
Scatchard plot (Fig. 5C) indicated a ratio of
high to low affinity sites of 1.17, a value that matched the
subunit stoichiometry previously
found(2) .
cc surface than to either of the
homomeric complex surfaces. In addition, Scatchard analysis (Fig. 5D) confirms the presence of two binding sites. The K
values for both sites
were in agreement with the other K
values
obtained for this analog at each site (). Unlike
T41P, the D20K analog appeared to bind to the
cc
surface at a single site. The kinetic constants ()
and the K
() were very similar to the values obtained
from binding to the
cc surface. These results are in
accordance with the observation that this mutation disrupts
-subunit binding (no binding was detected to the
cc
surface), and therefore binding to the
cc surface is
approximately equivalent to the
cc surface. It should be
noted that the K
value
(108 nM) was about 3-fold greater than obtained for the
consensus K
values to the
cc
for this analog. Since this was the single instance where these values
did not closely correspond, it may reflect some small contribution of
the
-subunit to D20K binding on the
cc surface.
cc,
cc, and
cc surfaces.
Immobilization of these complexes proved very efficient using standard
amine coupling chemistry, presumably due to the presence of the
extended, lysine-rich coiled-coil domain. In addition, these surfaces
displayed a high ligand binding capacity and were extremely stable to
regeneration, suggesting that attachment through the coiled-coil stalk
resulted in little steric interference with the binding sites.
site () were approximately the same as previously
reported (5) for the
pseudo high affinity cell
surface receptor (k
= 1.66
10
(M
s)
, k
= 1.11
10
s
). The equilibrium dissociation constants
reported in that study (K
= 0.67 nM and K
= 0.60
nM) also matched those in .
-subunit specific mutation (T41P) suffered greater than a
20-fold reduction in affinity to the
cc surface. In
comparison, the D20K analog carrying a
-subunit specific mutation
bound to the
cc surface in a fashion indistinguishable from
IL-2. This analog, however, showed no tendency to bind to the
cc surface, an observation consistent with the nature of the
mutation. On this surface, the T41P analog displayed properties similar
to IL-2. Binding of these analogs to the heteromeric
cc
surface provided further support for the conclusion that this complex
is composed of single high and low affinity sites. The T41P analog
bound to this surface with 80-100-fold higher affinity than to
either of the homomeric subunit surfaces, and Scatchard analysis of
binding confirmed the presence of two sites with 1:1 stoichiometry, as
was observed for IL-2 itself. This is consistent with the observation
that, although the proline exchange at position 41 greatly reduces
-subunit interaction, it does not completely eliminate
it(12) . The higher affinity of this analog to the
cc surface also suggests that the details of
IL-2
-subunit interaction may differ when bound to the
-subunit alone compared with the
pseudo high affinity
complex.
-subunit binding was more severe. This analog bound to the
cc surface as if the
-subunit was absent from the
complex. In fact, with respect to all binding parameters, the D20K
analog interacted with both the
cc and
cc
surfaces in a manner indistinguishable from the interaction of IL-2
with the
cc surface.
cc complexes interact with ligand in
a fashion that mimics the comparable cell surface receptors but also
demonstrate the utility of SPR biosensor methodology in the ligand
binding analysis of complex cytokine receptor systems.
- and
-receptor
subunit specific point mutations, respectively.
Table: Equilibrium dissociation constants
= k
/k
) in
Table I and from Scatchard analysis (K
) of the equilibrium
bound values R
for each concentration of ligand
injected as depicted in Fig. 5. NA refers to values that could not be
determined due to the inability to accurately measure the kinetic
constants or lack of detectable binding. Values for K
in parentheses are
those determined for the low affinity
site in the
cc complex. T41P and D20K are IL-2 analogs with
-
and
-receptor subunit specific point mutations, respectively.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.