From the
Human interleukin 5 (hIL5) and soluble forms of its receptor
Interleukin 5 (IL5)
Interleukin 5 exerts its biological effects via cell
surface receptor proteins (Chihara et al., 1990; Lopez et
al., 1990; Plaetinck et al., 1990; Migita et
al., 1991). The human IL5 receptor is composed of two types of
subunits, denoted
The ligand binding of human IL5
receptor
The stoichiometry of IL5 binding to
We have initiated an investigation of the quantitative and
structural properties of the interaction of hIL5 with its receptor. In
this report, we describe the expression and production of
Drosophila-expressed hIL5 and both soluble and
membrane-associated forms of its receptor
Materials For protein purification, Q-Sepharose fast flow, phenyl-Sepharose fast
flow high substitution, Superose 12 and 6, and Superdex 75 were from
Pharmacia LKB Biotechnol. Hydroxylapatite high resolution medium was
from Calbiochem. ABX-plus ion exchange resin was from J. T. Baker
Chemical Co. Lentil lectin-agarose and methyl
D-mannosylpyranoside were from E-Y Lab (San Mateo, CA). For
BIAcore kinetics measurements, the sensor chips CM5, surfactant P20,
and the amine coupling kit containing N-hydroxysuccinimide
(NHS), N-ethyl- N`-(3-diethylaminopropyl)carbodiimide
(EDC), and ethanolamine hydrochloride all were from Pharmacia
Biosensor. Murine IL3 was obtained from Genzyme (Cambridge, MA), and
purified murine IL5 (specific activity 4.9
Electrospray mass spectra were
recorded on a Sciex API-III triple quadrupole mass spectrometer fitted
with a standard pneumatically assisted nebulization probe and an
atmospheric pressure ionization source (Sciex, Ontario, Canada).
Buffers and salts were removed by high performance liquid
chromatography prior to mass spectrometry. Sample was loaded in aqueous
buffer onto a 2.1 mm inner diameter C8 guard column, flushed with 0.1%
trifluoroacetic acid for 5 min and then step eluted with 60%
CH
Cross-linking was performed by
incubating 1 n
M each of
X-ray diffraction data were measured from a single
crystal using a Siemens two-dimensional position-sensitive detector.
Approximately 5776 reflections were measured in 2 days to give 3054
unique reflections to 2.6-Å resolution, representing 84% of the
data. The merging R factor for symmetry-related reflections
was 0.042.
The structure of the deglycosylated hIL5 monomer was
determined by molecular replacement using XPLOR (Brunger, 1992). The
starting model consisted of all atoms of the dimer from the previously
reported crystal structure of hIL5 (Milburn et al., 1993).
Rotation functions were calculated using x-ray diffraction data from 10
to 5 Å and a radius of integration of 27 Å. Results from
the cross-rotation function revealed two peaks
(
The monomer was refined using XPLOR. The
resulting phases were used to calculate Fourier maps with coefficients
Sedimentation equilibrium
data were analyzed using nonlinear least-squares methods (Johnson and
Frasier, 1985; Brooks, et al., 1994a, 1994b) under the control
of a modified version of IGOR-PRO (Wavemetrics, Lake Oswego, OR)
running on an Apple MacIntosh computer. Data sets were collected after
reaching equilibrium, usually about 18 h, at indicated rotor speeds.
Equilibrium was established by determining that scans taken 4 h apart
were superimposible.
At equilibrium, the concentration distribution
of a single, homogeneous species is given by
On-line formulae not verified for accuracy
Kinetic data were evaluated using relationships
described pre-viously (Karlsson et al., 1991; Morton et
al., 1994). For the bimolecular interaction,
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Structural Analysis and Sedimentation Behavior of Recombinant
Proteins
The molecular mass
estimated for shIL5R
An IL5- and IL3-dependent B cell proliferation assay was used to
verify the functional activity of the recombinant proteins.
Drosophila-expressed hIL5 caused the proliferation of murine
B
The adequacy of Drosophila-specific glycosylation
for binding activity was assessed by enzymatic deglycosylation of
shIL5R
Crystals of deglycosylated hIL5 were grown from 27% PEG-600 or
PEG-1000 in 100 m
M Tris buffer, pH 8.0, at 25 °C. The
structure determined using these crystals is essentially identical to
that previously reported by Milburn et al. (1993) for E.
coli-expressed hIL5, except in two regions (Fig. 3). These are the
NH
A species analysis of sedimentation equilibrium data (see
``Experimental Procedures'') was used to examine the
stoichiometry of the hIL5
Needle crystals were grown from a mixture of
Drosophila-expressed hIL5 and shIL5R
We analyzed the contents of the
cocrystals for the abundance of both putative components of the
hIL5
We examined further aspects of the equilibrium of the
hIL5
The thermodynamics for hIL5 binding to
shIL5R
The observed
molar ratio of hIL5 bound to shIL5R
Fig. 7
shows the temperature dependence of the enthalpy change for
the reaction of hIL5 with shIL5R
The kinetics of binding of hIL5 to shIL5R
In a previous study, analysis of
data from the early or faster phases of the association (excluding the
very early phase which likely is dominated by mass transport) and
dissociation processes yielded k
The affinity of hIL5 binding to membrane-bound receptor was examined
using the full-length
The
Drosophila expression system also allowed the production of
cells containing full-length receptor incorporated into the
extracellular membrane surface. The binding affinity of soluble and
full-length receptor could be compared, with the expectation that the
carbohydrate structures would be similar for these two
Drosophila-expressed forms of the receptor and hence that the
only difference would be the presence of the membrane spanning region
and cytoplasmic tail in the full-length form versus their
absence in the soluble form.
The similarity
of affinities for hIL5 binding to both soluble and membrane-bound
(full-length)
The data reported here confirm
that the affinity of hIL5 for its recognition receptor subunit,
The total surface area buried in
the binding reaction of hIL5 and its soluble receptor
On-line formulae not verified for accuracy
The very large value of 4160
Å
Further analysis of the possibility that
conformational changes are coupled to the IL5-shIL5R
On-line formulae not verified for accuracy
The multiphasic association and dissociation steps
observed in the current study in biosensor kinetic analysis of
hIL5
We are grateful to Dr. Steven R. Jordan (Glaxo,
Research Triangle Park, NC) for atomic coordinates of E.
coli-expressed human IL5 and to Dr. Kalyan Anumula (at SmithKline
Beecham) for his assistance in carbohydrate analysis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit were expressed in Drosophila cells and purified
to homogeneity, allowing a detailed structural and functional analysis.
B cell proliferation confirmed that the hIL5 was biologically active.
Deglycosylated hIL5 remained active, while similarly deglycosylated
receptor
subunit lost activity. The crystal structure of the
deglycosylated hIL5 was determined to 2.6-Å resolution and found
to be similar to that of the protein produced in Escherichia
coli. Human IL5 was shown by analytical ultracentrifugation to
form a 1:1 complex with the soluble domain of the hIL5 receptor
subunit (shIL5R
). Additionally, the relative abundance of ligand
and receptor in the hIL5
shIL5R
complex was determined to be
1:1 by both titration calorimetry and SDS-polyacrylamide gel
electrophoresis analysis of dissolved cocrystals of the complex.
Titration microcalorimetry yielded equilibrium dissociation constants
of 3.1 and 2.0 n
M, respectively, for the binding of hIL5 to
shIL5R
and to a chimeric form of the receptor containing
shIL5R
fused to the immunoglobulin Fc domain (shIL5R
-Fc).
Analysis of the binding thermodynamics of IL5 and its soluble receptor
indicates that conformational changes are coupled to the binding
reaction. Kinetic analysis using surface plasmon resonance yielded data
consistent with the K
values from
calorimetry and also with the possibility of conformational
isomerization in the interaction of hIL5 with the receptor
subunit. Using a radioligand binding assay, the affinity of hIL5 with
full-length hIL5R
in Drosophila membranes was found to be
6 n
M, in accord with the affinities measured for the soluble
receptor forms. Hence, most of the binding energy of the
receptor
is supplied by the soluble domain. Taken with other aspects of hIL5
structure and biological activity, the data obtained allow a prediction
for how 1:1 stoichiometry and conformational change can lead to the
formation of hIL5
receptor
complex and signal
transduction.
(
)
is a
disulfide-linked, glycosylated dimeric protein which plays a prominent
role in the maturation, proliferation, and activation of eosinophils
(Basten and Beeson, 1970; Metcalf et al., 1974; Warren and
Sanderson, 1985; Sanderson et al., 1985; McKenzie and
Sanderson, 1992; Campbell et al., 1987; Clutterbuck et
al., 1987; Lopez et al., 1988). IL5's central role
in the control of eosinophilia has suggested that it is a major cause
of tissue damage in asthma and other eosinophil-related disorders
(Hamid et al., 1991; Bentley et al., 1992; Corrigan
and Kay, 1992).
and
(Tavernier et al.,1991). The cloned cDNA for the
subunit (Murata et
al., 1992) encodes a glycoprotein of 420 amino acids with an
amino-terminal hydrophobic region (signal sequence, 20 amino acid
residues), a glycosylated extracellular domain (324 residues), a
transmembrane domain (21 residues), and a cytoplasmic domain (55
residues). The
subunit expressed in a soluble form (the
extracellular domain) can bind to IL5 without the
chain and is
IL5-specific (Tavernier et al., 1991; Murata et al.,
1992; Devos et al., 1993). In contrast, the
chain is
identical to the
chains of GM-CSF and IL3 receptors (Tavernier
et al., 1991; Lopez et al., 1990) and appears to be
needed for signal transduction.
chain (hIL5R
) is of high affinity, with a
K
of 0.6-1 n
M reported for
the
subunit expressed in various heterologous cells (Tavernier
et al., 1991, 1992; Takaki et al., 1993). The above
studies showed that the affinity for
subunit alone was lower by a
factor of 2-4-fold than that for cells transfected with both
and
subunits. Direct interaction of the
subunit with
IL5 has been suggested by cross-linking (Devos et al., 1991;
Tavernier et al., 1991, 1992; Takaki et al., 1993)
and as such could contribute energetically to the IL5 receptor
affinity.
subunit has been
suggested to be 1:1 (one
subunit/IL5 dimer). This was based on
both cross-linking data as well as from quantitating the abundance of
each component in a complex isolated by immunoaffinity capture followed
by gel filtration chromatography (Devos et al., 1993). A
1:1-sized cross-linked IL5-receptor
chain complex also has been
observed indirectly (Mita et al., 1988; Migita et
al., 1991; Devos et al., 1991; Tavernier et al.,
1992). The above 1:1 stoichiometry is unexpected, since the IL5 dimer
has two 4-helix bundle domains. Each of these domains resembles the
4-helix bundle domain of monomeric growth factor proteins such as
growth hormone (Abdel-Meguid et al., 1987; de Vos et
al., 1992) and IL4 (Redfield et al., 1991), and each of
the latter can bind at least one molecule of receptor/molecule ligand.
Each IL5 monomeric domain might therefore be expected to bind at least
one receptor molecule. Interestingly, the molecular weight of the
cross-linked product of IL5 with
chain also suggests a 1:1
stoichiometry (Devos et al., 1991; Tavernier et al.,
1992). The structural basis for the 1:1 stoichiometry of IL5 and its
and
receptor subunits is as yet undetermined. The above
methods used to define 1:1 stoichiometry, namely cross-linking and
chromatographic isolation of complexes, likely would not detect weakly
associating multimeric complexes. Nonetheless, if correct, 1:1
stoichiometry must be accounted for in explaining signal transduction.
chain. We report the
three-dimensional structure of deglycosylated
Drosophila-expressed hIL5 and show that it is nearly identical
to the protein produced in Escherichia coli (Milburn et
al., 1993). We further report the interaction properties of these
functionally active proteins. The equilibrium binding parameters of
soluble and full-length IL5R
chain show that the soluble domain of
the receptor accounts for most, if not all, of the binding energy of
cell-bound receptor
subunit recognition. We provide rigorous
confirmatory evidence for a stoichiometry of 1:1 for the
hIL5
receptor
chain complex. Finally, we report new data on
the thermodynamics and kinetics of the IL5
receptor interaction
which argue that conformational change occurs in this process.
Together, the data suggest a model for the interaction of hIL5 with its
receptor
and
subunits leading to signal transduction.
10
units/mg) was prepared in a baculovirus expression system using
Spodoptera frugiperda 21 cells (Mitchell et al.,
1993). The murine pre-B cell line, denoted B13, was obtained courtesy
of R. Palacios, Basel Institute of Immunology, Switzerland. Expression of Recombinant IL5, shIL5R
, and shIL5R
-Fc Human IL5 and various forms of shIL5R
were expressed in a
Drosophila cell culture system (van der Straten et
al., 1989; Angelichio et al., 1991). Each gene to be
expressed was cloned between a copper sulfate-inducible metallothionein
promoter and an SV40 late polyadenylation site and introduced into
Drosophila Schneider 2 (S2) cells by cotransfection with a
vector carrying a hygromycin B resistance gene. Hygromycin B-resistant
cells were selected as a stable polyclonal population, induced with
copper sulfate, and examined for expression by Western blot analysis.
Antisera used for Western blots were raised by immunization of rabbits
with denatured fusion proteins (expressed in E. coli and
gel-purified) carrying hIL5 or hIL5R
sequences.
hIL5 Expression
Human IL5 expression was achieved with a
vector encoding a fusion of a human tissue plasminogen activator (tPA)
secretion signal sequence to amino acids 4-115 of mature hIL5.
Cleavage of the signal sequence during secretion leaves four amino
acids from tPA fused to the NHterminus of hIL5, as
reported for other such fusions (Culp et al., 1991). Western
blot analysis of medium from induced cultures revealed a protein with
an apparent molecular mass of 15 kDa under reducing conditions and 30
kDa under nonreducing conditions, as expected for the disulfide-linked
homodimer. The cell line was expanded to 30 liters to produce hIL5.
shIL5R
To express shIL5R Expression
in
Drosophila, a cDNA fragment encoding the shIL5
protein
precursor (signal sequence followed by mature shIL5R
) was cloned
from human eosinophil RNA by polymerase chain reaction and inserted
into an expression vector (pMTAL) to yield pMTAL-shIL5R
, a vector
that does not contain the tPA sequence. DNA sequence analysis showed
the shIL5R
sequence to be identical to that reported by others
(Tavernier et al., 1992). Western blot analysis of induced
culture carrying this construct revealed a broad band with an apparent
molecular mass of 40-43 kDa under both reducing and nonreducing
conditions. The cell line was expanded to 4 liters in spinner flasks
for production of shIL5R
.
shIL5R
A fusion of
shIL5R-Fc Chimera Expression
to the C
2-C
3 region of human IgG1
was constructed and then expressed in Drosophila cells as
described above for shIL5R
. In this construct, the COOH terminus
of shIL5R
(Arg
of mature shIL5R
) was linked to
the NH
terminus of the IgG1 hinge region
(Glu
, Kabat numbering) by the short sequence
Ile-Glu-Gly-Arg, a Factor Xa cleavage site, allowing recovery of
shIL5R
by proteolytic cleavage of the purified chimera. The Fc
region also had a mutation of Cys
to Ala. Western
analysis using anti-shIL5R
antiserum showed that this protein (70
kDa under reducing conditions, 140 kDa and hence dimeric under
nonreducing conditions) was produced in Drosophila cell
cultures at levels comparable to shIL5R
lacking the Fc region. The
cell line was expanded to 4 liters for production of the Fc receptor
chimera. In the present study, we evaluated the binding properties of
the uncleaved and unreduced chimera (molecular mass 140 kDa), which
retains both sIL5R
fragments/molecule. Expression of Full-length hIL5R
in Drosophila Cells The transmembrane and cytoplasmic region of hIL5R
was cloned by
reverse transcriptase polymerase chain reaction from a butyrate-induced
eosinophilic subline of human promyelocytic HL-60 cells and the
intended DNA sequence confirmed. This fragment was cloned into
pMTAL-shIL5R
to create a new vector, pMTAL-hIL5R
, containing
the full-length receptor (20 amino acid signal sequence followed by 400
amino acid mature protein). Western blot analysis of induced cells
carrying this construct revealed a band with an apparent molecular mass
of 50 kDa. Protein Purification Expression levels of hIL5, shIL5R
, and shIL5R
-Fc in Drosophila media used for large scale purification were estimated to be 22,
17, and 10 mg/liter, respectively, from Western blot analysis and
Coomassie Blue staining of SDS-PAGE. Western blot analyses of all three
products in culture media suggested heterogeneity in the carbohydrate
content. Protein concentration of the starting material and at various
stages of purification was estimated by the Micro BCA Protein assay.
Extinction coefficients at 280 nm calculated from tryptophan and
tyrosine contents matched well with those measured by amino acid
analysis: E
(cm - mg/ml)
= 0.63 for hIL5, 1.9 for shIL5R
, and 1.65 for
shIL5R
-Fc. Throughout this study, concentrations of purified
proteins were based on absorbance at 280 nm.
Purification of hIL5
28 liters of sterile-filtered
Drosophila media were diluted 3-fold with water, adjusted to
pH 8.0, and loaded onto a Q-Sepharose FF column (11.4 14.1 cm,
Pharmacia) equilibrated with 20 m
M Tris-HCl, pH 8.0. hIL5 in
the flow-through fractions was pooled, adjusted to pH 7.4 with 6
M HCl, and loaded onto a hydroxylapatite column (HA) equilibrated
with Buffer T (20 m
M Tris-HCl, pH 7.4). The column was washed
with Buffer T, and bound hIL5 was eluted with 0.25
M potassium
phosphate, pH 7.5. Pooled fractions from the HA column were mixed with
an equal volume of 3
M ammonium sulfate and applied to
phenyl-Sepharose ff (5.7
7 cm) equilibrated with 1.5
M NH
SO
in Buffer P (20 m
M sodium
phosphate, pH 7.0). hIL5 was eluted with a linear gradient of
1.5-0
M NH
SO
in Buffer P. The
pooled fraction from phenyl-Sepharose (600 ml) was concentrated to 80
ml, loaded onto Superose 12, and eluted with Buffer T + 0.15
M NaCl. The final yield of hIL5, approximately 500 mg, was >98%
electrophoretically homogeneous based on silver staining of SDS-PAGE
gels; nucleic acid and endotoxin were undetectable.
Purification of shIL5R
4.3 liters of
sterile-filtered conditioned Drosophila media (MRD3) were
diluted 2-fold with Buffer T, adjusted to pH 7.4, and loaded onto a
Q-Sepharose column (5 8.8 cm, Pharmacia) equilibrated with
Buffer T. Receptor in flow-through fractions (9 L) was concentrated to
1900 ml using a Miniset 10K cutoff membrane (Filtron, Northborough MA).
The concentrated Q-Sepharose pool was applied to an ABX-plus column (5
7 cm, J. T. Baker Chemical Co.) equilibrated with Buffer A (20
m
M sodium acetate, pH 5.5), and washed with Buffer A. Bound
receptor was eluted with a linear gradient of 0-1
M NaCl
in Buffer A. Fractions containing receptor were immediately neutralized
with pH 7.4 1
M Tris-HCl buffer. This ABX pool (240 ml) was
adjusted to 5 m
M CaCl
, sterile filtered, and
loaded onto two lentil lectin columns (2.5
5.8 cm) in tandem,
equilibrated with Buffer C (20 m
M Tris-HCl, pH 7.2, 0.15
M NaCl, and 5 m
M CaCl
). Bound receptor was
eluted with 0.25
M methyl
D-mannosylpyranoside in
Buffer C. The lentil column pool (88 ml) was concentrated to 12 ml on a
YM-10 membrane (Amicon) and loaded onto a Superdex 75 preparation grade
column (2.6
96.4 cm) equilibrated in 0.1
M HEPES, pH
7.5. Pooled fractions (29 ml) were stored at -70 °C. The
Superdex 75 column eluate contained 20 mg of receptor, with >98%
electrophoretic homogeneity based on silver staining of SDS-PAGE gels.
Purification of shIL5R
4 liters of
conditioned Drosophila media were diafiltered into 0.1
M Tris-HCl, pH 8.1, and concentrated to 600 ml. The diafiltered and
concentrated media were loaded onto a Protein A-Sepharose ff column
(2.5 -Fc Chimera
5.8 cm, Pharmacia) equilibrated with 0.1
M Tris-HCl, pH 8.0, and washed with the same buffer. The Fc chimera
was eluted with 0.1
M glycine HCl, pH 3.0, and fractions were
collected in 1
M Trizma base to raise the final pH to 7.7. The
chimera was fractionated on a Superose 6 column into 0.1
M HEPES, pH 7.5. 2.8 mg of Fc chimera (>95% electrophoretically
homogeneous in SDS-PAGE) was obtained from 4 liters of media. Mass Spectroscopy Matrix-assisted laser desorption mass spectrometry (MALD-MS) data were
obtained on a Vestec Research model mass spectrometer (Persceptive
Biosystems, Boston, MA). Samples were prepared for analysis by mixing 1
µl of a 1-6 pmol/ml solution of protein in phosphate-buffered
saline, pH 7.4, with 1 µl of the matrix, sinapinic acid, on the
stainless steel target. The 337-nm line from a nitrogen laser (10 ns
pulse width, 10 Hz repetition rate) was used for desorption/ionization
of the sample. The spectra were the sum of
20-70 laser
shots. Spectra were calibrated externally using the BSA (bovine serum
albumin) and BSA dimer peaks.
CN, 40% H
O, 0.1% trifluoroacetic acid. An
aliquot of the sample was concentrated to near dryness and brought to a
final concentration of
20 pmol/µl with methanol/water (50:50
v/v) containing 0.2% formic acid. Sample was introduced into the mass
spectrometer by infusion with a syringe pump (Harvard Instruments) at 2
µl/min. Approximately 2 min of data were recorded and
40 pmol
of the sample consumed. Enzymatic Deglycosylation and Cross-linking to
I-hIL5 hIL5 was iodinated with Bolton-Hunter reagent to a specific activity of
>350 Ci/mmol using the manufacturer's protocol (DuPont NEN).
100 µg of shIL5R
or 10 µg of
I-hIL5 were
enzymatically deglycosylated by treating with 5,000 units of PNGaseF
(New England Biolabs) in 50 m
M sodium phosphate, pH 7.5, for
12-16 h at 37 °C in the presence of a mixture of protease
inhibitors (1 m
M phenylmethylsulfonyl fluoride, 2 µg/ml
aprotinin, 20 µ
M leupeptin). Analysis of the shIL5R
product by Western blot showed a reduction in molecular mass of
3-4 kDa relative to untreated receptor, consistent with loss
of carbohydrate but absent proteolytic degradation (data not shown).
The mobility was identical to shIL5R
enzymatically deglycosylated
in denaturing conditions (0.5% SDS, 1%
-mercaptoethanol),
suggesting that deglycosylation was complete. Similarly,
deglycosylation of hIL5 led to
1 kDa reduction in mobility on
Western blots (data not shown).
I-hIL5 and shIL5R
together with 1 n
M of the bifunctional cross-linker
bis-suberimidate (Pierce) in 25 m
M HEPES, pH 7.2, 0.1% BSA for
20-30 min at 4 °C. Products were analyzed by 12.5% SDS-PAGE
in reducing conditions, followed by gel drying and autoradiography. B13 Cell Proliferation Assay The murine IL5/IL3-dependent cell line LyH7.B13 was subcultured twice
weekly in RPMI 1640 medium (Life Technologies, Inc.), supplemented with
L-glutamine, non-essential amino acids, sodium pyruvate,
penicillin-streptomycin (all Life Technologies, Inc.), plus
2-mercaptoethanol (5
10
M, Sigma),
10% fetal bovine serum (Globepharm), and 1-10 units of
recombinant murine IL5 (Mitchell et al., 1993). For assays,
cells were washed and maintained in medium without IL5 for a minimum of
2 h. They were cultured for 48 h in triplicate (5,000 cells/well) in
96-well round bottom plates in the presence of appropriately diluted
test samples and pulsed with 0.5 µCi of
[
H]thymidine (Amersham) for the final 4 h. They
were processed for scintillation counting in a 1205 Betaplate (LKB
Wallac). Results are presented as Stimulation Index ± standard
deviation, relative to unstimulated signal. Crystallization Crystals of deglycosylated hIL5 (from PNGaseF treatment in 0.1
M HEPES, pH 7.4, 25 °C, 48 h) and of the complex between
glycosylated hIL5 and shIL5R
were obtained using the hanging drop
method of vapor diffusion (McPherson, 1976). Deglycosylated hIL5 was
concentrated to approximately 10 mg/ml in 100 m
M HEPES buffer,
pH 7.2. The complex was prepared by mixing equimolar amounts of hIL5
(dimer) and shIL5R
in 100 m
M HEPES, pH 7.2. Typically, 2
µl of either protein solution were mixed with 2 µl of various
precipitant concentrations and vapor equilibrated at several
temperatures against the solution added to the protein. At each
temperature, a three-dimensional matrix was established for each
precipitant, in which protein concentration, precipitant concentration,
and pH were varied. Ammonium sulfate, sodium chloride, and various
polyethylene glycols were used as precipitants. Determination of Molecular Ratio of hIL5 and shIL5R
in Cocrystals Cocrystals were harvested under a microscope, washed twice in
crystallization solution P (27% PEG 600 (v/v) in 100 m
M sodium
acetate, pH 5.0), solubilized in SDS-sample buffer, and analyzed by 12%
SDS-PAGE. The gels were either stained using Coomassie Blue or
silver-stained. Intensities of the bands of IL5 and shIL5R
in the
cocrystals were compared with those of controls (hIL5, shIL5R
, or
combinations of both proteins) by scanning with IS-1000 Digital Imaging
System (Alpha Innotech Co, San Leandro CA), and the peak areas were
integrated. Crystal Structure Determination of Deglycosylated hIL5 Crystals of deglycosylated hIL5 belong to the space group C2 with a = 118.3 Å, b = 24.3
Å, c = 43.8 Å, and
= 110°.
Assuming one deglycosylated monomer/asymmetric unit, the
V
value is 2.1
Å
/dalton, which is within the range found for most
protein crystals (Mathews, 1968). These crystals show diffraction
beyond 2.6-Å resolution and are stable in the x-ray beam for
several days.
,
,
are 166.9, 131.5, 159.3° and 345.7, 50.8, 200.7°) related
by a 2-fold axis. Translation was done manually on the graphics by
moving rotated dimer along the a and c axes, so that
the b (2-fold) axis passed through the center of the dimer.
Rigid body refinement of translated monomer gave an R factor
of 0.49 using 2.0
( F
) data from 8-
to 3- Å resolution.
F
-
F
and
2
F
-
F
,
into which the atomic model was analyzed. Four water molecules were
found and included in the model. The R factor was reduced to
0.231 when data greater than 2
( F
),
in the resolution range 8-2.6Å, were used to refine all
atomic positions of the monomer and their temperature factors. The
overall root-mean-square deviation from ideal geometry for bond lengths
was 0.009 Å, while that for bond angles was 1.69°. Analytical Ultracentrifugation Equilibrium sedimentation experiments were performed with a Beckman XL-A
analytical ultracentrifuge. Double sector cells with charcoal-filled
epon centerpieces and sapphire windows were used. Sedimentation
experiments were performed at 25.0 °C in 0.1
M HEPES, pH
7.4. The partial specific volumes of the monomers and the potential
complexes were calculated using the partial specific volumes of the
component amino acids (Cohn and Edsall, 1943), which yielded a value of
0.741 ml g
for IL5, 0.722 ml g
for shIL5R
, 0.730 ml g
for the 1:1 (molar
basis) complex, and 0.727 ml g
for 2:1 complex
(molar basis). The solvent density,
, was estimated to be 1.006 g
ml
. To test the hypothesis that IL5 could
potentially interact with 2 mol of shIL5R
, shIL5R
and IL5
were mixed in two ratios. For one, shIL5R
was in small excess of
IL5, on a molar basis. For the other, shIL5R
was in larger excess
of IL5, i.e. approximately 2 mol of shIL5R
/mol of IL5.
For both experiments the total protein concentration was
10
M.
Sequence Analysis
The amino acid compositions and
NH-terminal sequences of all three proteins agreed well
with those predicted from the cDNA sequences (Tanabe et al.,
1987; Tavernier et al., 1991). For IL5 the
NH
-terminal sequence was NH
-GARSEIPTSALVKET.
The GARS sequence was from the TPA leader sequence used in
expression (see ``Experimental Procedures''). The
COOH-terminal peptide, produced by CNBr cleavage, was
NTEWIIES-CO
H. The NH
-terminal sequences of
shIL5R
and shIL5R-Fc chimera were
NH
-DLLPDEKISLLPV- x, where x is a
potential site of N-linked glycosylation.
Molecular Mass
Molecular masses of the
Drosophila-expressed recombinant proteins were estimated by
SDS-PAGE as well as by gel filtration. Human IL5 migrates by SDS-PAGE
as a single band at 32 kDa under non-reducing conditions and 16 kDa
under reducing conditions (dithiothreitol, see Fig. 1 A),
confirming that the Drosophila hIL5 is a disulfide-linked
dimer. Human IL5 was eluted from Superose 12 as a dimer (data not
shown). The molecular mass of hIL5 was measured by electrospray-mass
spectrometry as 28,492 ± 2 Da (data not shown). Of this, 26,418
Da was calculated to have originated from the amino acid residues
(Murata et al., 1992) and the remaining from carbohydrate.
This mass difference fits precisely the combined residue masses of two
N-linked ManGlcNAc
Fuc moieties and is
constant with carbohydrate structures expressed in insect cell lines
(see Carbohydrate Analysis, below) (Hsieh and Robbins, 1984; Kuroda,
et al., 1990). A global analysis of equilibrium sedimentation
data from three rotor speeds yielded a single molecular mass of 28.1
± 0.4 kDa and demonstrated that hIL5 was a dimer in solution and
did not aggregate further (Fig. 2 A).
(315 residues, 35,870 Da) was 46 kDa by
non-reducing SDS-PAGE (Fig. 1 A) and 44 kDa by size
exclusion chromatography (data not shown), indicating that shIL5R
was monomeric, in spite of the presence of 1 unpaired cysteine residue.
A molecular mass of 42 kDa was determined by MALD-MS (data not shown).
From these values, it is evident that
14% of the mass of
shIL5R
was carbohydrate. A global analysis of equilibrium
sedimentation yielded a single molecular mass of 44 ± 0.8 kDa
(Fig. 2 B). There appeared to be some tendency for shIL5R
to self-associate to a tetrameric form at higher protein
concentrations. The monomer was 50% associated at 1.0
10
M (data not shown). With this affinity,
the receptor was <1% associated at 10
M.
Figure 1:
SDS-PAGE analysis of Drosophila expressed recombinant proteins. A, purified soluble
proteins, 20 µg, 12% gel stained with Coomassie blue: hIL5
( lanes 4 and 7), shIL5R ( lanes 3 and
6), and shIL5R
-Fc ( lanes 2 and 5).
Protein standards are in lanes 1 and 8. Lanes
1-4 were run under nonreducing conditions and lanes
5-8 under reducing conditions. B, full-length
hIL5R
on Drosophila cells, gel stained by Western
blotting. Protein was detected using a rabbit antiserum raised against
an E. coli expressed fusion protein, containing residues
196-293 of mature sIL5R
following 81 amino acids of
influenza virus structural protein NS1 (Shatzman et al.,
1990). Samples are: lane 1, pMT-shIL5R
culture medium
from induced cells; lane 2, pMT-hIL5R
cells uninduced;
and lane 3, pMT-hIL5R
cells
induced.
shIL5R-Fc (553 residues, 62,542 Da) migrated by SDS-PAGE at
67.7 kDa under reducing conditions and 145 kDa under non-reducing
conditions (Fig. 1 A), indicating the molecule was a
disulfide-linked immunoglobulin-like dimer. It eluted from Superose 6
with an apparent mass of 178 kDa (data not shown). MALD-MS showed a
mass of 140,280 Da, indicating that 11% of the mass was from
carbohydrate (data not shown). Analytical ultracentrifuge analysis
showed that shIL5R
-Fc had a tendency to associate at higher
concentrations. These data were best fit to a monomer-tetramer
equilibrium, with 50% tetramer formation at 6
10
M, under these solvent conditions. With this affinity,
the shIL5R
-Fc was <4% associated at 10
M. Thus, self-association of receptor was concluded not
to be a complicating factor in the subsequent biophysical experiments.
Carbohydrate Analysis
Monosaccharide composition
was determined as described previously (Anumula and Taylor, 1989).
Oligosaccharide map analysis indicated that the major oligosaccharide
of both proteins had the structure of
ManGlcNAc
Fuc (data not shown). The high mannose
content, lack of sialic acid, and presence of two glucosamines and a
fucose at each glycosylation site are typical for proteins expressed in
Drosophila (Culp et al., 1991). Bioactivity of Recombinant hIL5 and Receptor Proteins
cells (specific activity = 1.7
10
units/mg), consistent with the cross-recognition of hIL5 by the
murine receptor. The level of activity obtained with
Drosophila-expressed protein was similar to that seen with
native hIL5 produced by peripheral blood T lymphocytes stimulated with
mitogen. Soluble hIL5R
was able to antagonize the activity of hIL5
in the same B
assay, with an IC
of 12
n
M. In contrast, the response elicited by murine IL3 was
unaffected by shIL5R
at concentrations up to 40 n
M.
Anti-IL3 antibody, on the other hand, completely inhibited the response
to IL3.
and assay by cross-linking to
I-hIL5. As
expected from previous data (Proudfoot et al., 1990),
PNGaseF-treated
I-hIL5 produced a 1:1 cross-linked
product with untreated shIL5R
. However, using similar enzymatic
deglycosylation with PNGaseF, untreated
I-hIL5 could not
be cross-linked to deglycosylated shIL5R
(data not shown). The
results suggest that receptor glycosylation, although not hIL5
glycosylation, is required to stabilize receptor-ligand interaction. Crystal Structure of Deglycosylated hIL5
-terminal helix and the loop between helix 2 and helix 3.
Superposition of the 216
-carbon atoms from the two structures
gives an root-meam-square deviation of 1.6 Å, while superposition
of 1,742 backbone and side chain atoms gives an root-mean-square
deviation of 2.1 Å. Overall, the crystal structure of
deglycosylated, Drosophila-expressed hIL5 confirms its basic
structure as a covalent dimer of two 4-helix bundles, similar to that
determined before for E. coli-expressed hIL5 (Milburn et
al., 1993). Determination of Stoichiometry of IL-5
shIL5R
Complex by
Analytical Ultracentrifugation
receptor interaction. The results of
analyses for two different mixtures of shIL5R
and IL5 are given in
Fig. 4, A and B. In both cases, the data fit best to
a mixture of the two components, hIL5 receptor (44.0 kDa) and the 1:1
complex of shIL5R
and IL5 homodimer (72.1 kDa). There is no
evidence from either data set for the presence of a 2:1 complex (116.1
kDa) or free IL5 (28.1 kDa). These data show that shIL5R
and IL5
(dimer) associate in solution as a 1:1 molar complex at concentrations
up to 40 µ
M. The data show no evidence of weak complexes,
which likely would be observed under the conditions of the
ultracentrifuge experiments if they had K
values of 0.1 m
M or less. Cocrystallization of hIL5 and shIL5R
and Cocrystal Analysis
in the presence of
24% PEG 1000 (v/v), 100 m
M sodium acetate/acetic acid, pH 5.0,
incubated at 7 °C. Slightly larger crystals were grown from 27% PEG
600 (v/v) in the same buffer.
shIL5R
complex by thoroughly washing and dissolving them
as described under ``Experimental Procedures'' and then
examining their contents by SDS-PAGE. The gel scan of the
Coomassie-stained gel is shown in Fig. 5. Gel scanning along with
standards containing various ratios of the two components showed that
the ratio of peaks of hIL5 to shIL5R
in the cocrystals was 1.39,
compared to a ratio of 1.26 for an equimolar mixture of the two
proteins. In silver-stained SDS-PAGE, the ratio for the equimolar
mixture was 1.58 while that of cocrystal was 1.59. These results
indicate that the stoichiometric ratio of hIL5 dimer versus shIL5R
in the cocrystal is 1:1, consistent with the results
obtained by analytical ultracentrifugation. Equilibrium Binding Properties by Titration Microcalorimetry
receptor interaction using titration calorimetry, particularly
to obtain a measure of the equilibrium affinity of the solution
interaction. Fig. 6 shows representative titration microcalorimetric
data for the equilibrium binding reaction of hIL5 with its soluble
receptor. Data were fit according to a single-site binding model.
Titration experiments were carried out as a function of temperature in
order to determine the heat capacity change for the reaction of hIL5
with shIL5R
. High quality data were obtained from 17 to 35 °C.
Data could not be measured at lower temperatures due to the decrease in
signal-to-noise with temperature. This is a consequence of the heat
capacity change of the reaction. High temperature data could not be
measured either, presumably due to thermal inactivation of shIL5R
.
At 45 °C no heat of binding could be detected, suggesting the
shIL5R
was unfolded.
and shIL5R
-Fc are summarized in Table I. The tabulated
K
values at 25 °C are based on
measurements of K
values at higher
temperature where the affinity was a little weaker, and therefore
somewhat easier to measure. The K
value
for IL5 binding to sIL5R
was measured as 5.9 n
M at 33
°C, and the K
value for IL5 binding
to sIL5R
-Fc was measured as 3.2 n
M at 35 °C. Both
measurements were done in triplicate and gave results within a factor
of two of the reported values. These experimental
K
values were corrected to 25 °C
according to the known temperature dependence (Robert et al.,
1989) using enthalpy and heat capacity changes determined from analysis
of the data in Fig. 7. The error associated with any single measurement
of the K
was about a factor of 2-3,
due to the relatively high affinity of the binding interaction and the
small enthalpy change for the reaction. Nevertheless, repeat
measurements of the K
consistently gave
values well within the factor of 2-3 error limit.
and shIL5R
-Fc was 0.98
± 0.03 and 1.61 ± 0.05, respectively. These values are
consistent with stoichiometries of 1:1 and 2:1 for hIL5 binding to
shIL5R
and shIL5R
-Fc, respectively. The higher stoichiometry
for the Fc chimera (molecular mass 140 kDa) is expected since there are
two receptor
chain domains/chimera. The molar ratio of 0.98 found
for shIL5R
is a demonstration of its high functional purity given
the 1:1 stoichiometry determined by analytical ultracentrifugation. On
the other hand, the ratio of 1.61 for the shIL5R
-Fc case suggests
that 20% of the potential binding sites were inactive.
and shIL5R
-Fc. The slope of
this temperature dependence is equal to the heat capacity change for
the equilibrium reaction and is due in large part to changes in buried
surface area of polar and apolar amino acid side chains (see
``Discussion''). Regression analysis of the data in
Fig. 7
gives the best-fit
H and
C
values for the reaction of hIL5
with shIL5R
and shIL5R
-Fc listed in . Correction
of the measured enthalpies for buffer ionization heats was not
necessary, by virtue of the low ionization heat of phosphate
(Christensen et al., 1976) and the pH independence of the
hIL5-shL5R
binding affinity in the neutral pH range (Morton et
al., 1994).
Figure 7:
Enthalpy change for IL-5 binding to
shIL5R and shIL5R
-Fc versus temperature. Circles are for shIL5R
and squares for shIL5R
-Fc. Slope
of the best-fit line yields the heat capacity change, which is
identical for the two receptor constructs, -0.65 kcal/mol
hIL5/degree. The heat capacity change for hIL5 binding is related to
the amount of surface area buried upon complexation with receptor (see
text). Each enthalpy change value was measured by titration calorimetry
as indicated in Fig. 6.
It can be seen from Fig. 7that the absolute
values of the IL5-binding enthalpies and the temperature dependences
for shIL5R and shIL5R
-Fc are, within error, identical. These
findings, in combination with affinity measurements (), are
strong evidence that the molecular details of the reactions of hIL5
with both types of soluble receptor constructs are the same.
Furthermore, the linearity of temperature dependence of the binding
enthalpy supports the simple binding model. Comparison of the tabulated
binding thermodynamics () for the interaction of soluble
receptor
with both the glycosylated or deglycosylated form of IL5
show the absence of any appreciable changes upon conversion to the
deglycosylated form. The data for the deglycosylated form were obtained
from a single measurement at 28 °C. A small temperature correction
to 25 °C was made by assuming that the
C
of the reaction is the same as it
is for the glycosylated hIL5. Kinetics and Equilibrium Binding Properties of hIL5 with Soluble
Receptor
from Optical Biosensor Analysis
-Fc were examined with
the receptor components immobilized on sensor chips of a BIAcore
biosensor. Sensorgrams and replots of the data according to Equations 5
and 6 are shown in Fig. 8. The linearizing replots for both the
association and dissociation processes showed nonlinearity
(Fig. 8, B and C). This type of curvilinearity
also is evident in the previously reported biosensor analysis of hIL5
interaction with shIL5R
(Morton et al., 1994). The
measured dissociation rate (slope of the plot in
Fig. 8C) is increased by including shIL5R
as a
competitor in the dissociation buffer, but the curvilinearity persists.
The 400 n
M concentration of competitor used in the
dissociation phase in Fig. 8 C is great enough (close to two
orders of magnitude greater) compared to K
that little or no rebinding is likely to occur under these
conditions. This result argues that the curvilinearity in the
dissociation phase is not due to rebinding.
Figure 8:
Kinetic analysis of on and off rates of
interaction of hIL5 with immobilized shIL5R-Fc. A,
sensorgrams showing binding of hIL5 (in response units), obtained at
hIL5 concentrations of 30, 20, 15, 10, 5, and 2 n
M, to
shIL5R
-Fc immobilized to the sensor chip via Protein A. For each
sensorgram, buffer was pumped over the sensor chip at 5 µl/min. At
100 s the buffer was replaced by the hIL5 solution. The increase in
response shows the binding of hIL5 from solution to the immobilized
receptor (the association phase). At 460 s the injection ended and was
replaced by buffer. The decay in response represents the dissociation
of bound hIL5 (the dissociation phase). B, association. The
association phases of the sensorgrams in A were replotted,
according to Equation 5, as the slope of the curve at a given time
( dR/dt) versus relative response at that time
( R). The replot is shown for the experiment with 27 n
M hIL5. The curvilinearity of this replot, typical of the data
obtained at all concentrations in particular at the five highest
concentrations, is not expected from Equation 4 for a single
exponential process. The dashed line is the linear fit to the
data from 235 to 295 s used to calculate k. Inset,
plot of k values (see ``Experimental Procedures,''
Equation 5) obtained at all concentrations. The k
value was determined from the slope of this plot. C,
dissociation. The dissociation phase of the sensorgram obtained at
highest hIL5 concentration in A (30 n
M) was replotted
as the log (response at start of dissociation/response at time n after dissociation) versus time (Equation 6). The data
shown are for dissociation after the injection of 30 n
M hIL5
on immobilized shIL5R
-Fc. A comparison also is shown of
dissociation phases for hIL5-shIL5R
-Fc interaction with and
without 400 n
M shIL5R
in dissociation buffer.
Curvilinearity of the plots in C is evident, suggesting that
dissociation does not behave according to Equation
4.
The curvilinearity of
both the association and dissociation data as shown in
Fig. 8
suggests that binding does not fit to a simple (A + B)
to AB binding model (Equation 4). The results could fit to a
conformational isomerization model as suggested by thermodynamics
analysis from titration calorimetry; this possibility is addressed
under ``Discussion.''
and
k
values, and consequent
K
values from k
and
k
, which fit with the K
obtained by steady state analysis (Morton et al., 1994).
Hence, in the current work, we estimated k
and
k
values from, respectively, the pseudolinear
portion of the association phase (from 175 to 255 s, corresponding to
187.2-299.7 response units, in the example shown in
Fig. 8B) and the first 50 s of the dissociation phase.
The average values obtained from two similar experiments (including
that in Fig. 8) were k
= 4.9
± 0.3
10
M
s
and k
= 3.7
± 0.1
10
s
. These
calculated values are taken as averages for the hIL5
shIL5R
-Fc
interaction in the sensor assay given the multiphasic nature of the
kinetics. The K
calculated from the rate
constant estimates is 7.6 ± 0.3 n
M, which is in
reasonable agreement with the titration calorimetry results. hIL5 Binding to Full-length
Chain Receptor in Drosophila Cells
chain expressed in Drosophila. As
shown in Fig. 9, iodinated hIL5 bound to Drosophila cell
membranes containing full-length hIL5R
, with the binding being
saturable and competed by unlabeled hIL5. Nonlinear regression analysis
of the binding data yielded a K
value of
6 n
M, a value within a factor of 2-3 of the values
determined by both titration calorimetry and biosensor kinetic
analysis. This agreement underscores that the soluble component of the
receptor
chain is sufficient for hIL5 recognition and that the
membrane-spanning and intracellular domains contribute relatively
little energetically to direct hIL5 interaction.
Drosophila Expression Yields Functionally Active
Recombinant hIL5 and hIL5 Receptor
In this study, we produced
large quantities of recombinant hIL5 and two forms of its receptor,
shIL5R and shIL5R
-Fc, in the Drosophila expression
system and used this material to determine interaction properties of
the hIL5
receptor complex. Drosophila-expressed proteins
(van der Straten et al., 1989; Angelichio et al.,
1991; Culp et al., 1991) are glycosylated, hence enabling
correlation of their properties with those of naturally glycosylated
proteins. Carbohydrate analysis of the hIL5 and receptor forms produced
here revealed molecular masses somewhat lower than their fully human
counterparts. Nonetheless, the Drosophila-derived hIL5 was
highly active in the B cell proliferation assay, and the
Drosophila-expressed shIL5R
and shIL5R
-Fc both
inhibited the cellular activity of the hIL5. Hence, the properties
determined for the Drosophila-expressed proteins can be
considered close reflections of those of fully human proteins.
The Stoichiometry of hIL5 and shIL5R
In this study, we confirmed that hIL5 dimer and shIL5R is
1:1
form a 1:1 complex. This was judged initially (data not reported) by
chemical cross-linking of
I-hIL5, an experiment similar
to that reported previously for COS- and baculovirus-expressed proteins
(Devos et al., 1993). We then obtained direct evidence of a
1:1 complex between hIL5 and its receptor by molecular weight analysis
of the complex using equilibrium sedimentation (Fig. 4).
Furthermore, both titration calorimetry experiments and analysis of
hIL5-shIL5R
cocrystals showed a 1:1 molar ratio for the reaction
of hIL5 with its
receptor. These data demonstrate the absence of
higher stoichiometries and aggregates for concentrations that approach
millimolar.
Figure 4:
Stoichiometry of hIL5 and shIL5R by
analytical ultracentrifugation. A, equilbrium analytical
ultracentrifuge data for a mixture of shIL5R
and hIL5, total
protein concentration
10
M, with a
small molar excess of shIL5R
over hIL5, as discussed under
``Experimental Procedures.'' Data obtained were fit to
Equation 3 in terms of the species which can be identified (see
``Experimental Procedures''). In this experiment, only terms
for shIL5R
and the shIL5R
hIL5 1:1 complex were needed to
fit the data. When terms for the other species, e.g. hIL5 and
shIL5R
hIL5 2:1 complex, were included, the fit did not
converge. B, a similar experiment to Fig. 5 A except
that initially shIL5R
was in larger excess to hIL5 (total protein
concentration
6
10
M).
The 1:1 stoichiometry for the hIL5receptor
interaction is unusual, given that the IL5 dimer has two 4-helix bundle
domains and each resembles the 4-helix bundle domain of monomeric
growth factor proteins (Abdel Meguid et al., 1987; Redfield
et al., 1991; de Vos et al., 1992). One might
therefore expect each IL5 4-helix bundle to bind at least one receptor
molecule. While the data here for 1:1 stoichiometry are for soluble
receptor, the observation of similar affinities of soluble and
full-length receptor (see below) suggests that hIL5 also binds to the
latter in the same 1:1 manner. By extrapolation from other cytokine
receptor systems, signal transduction by IL5 may require aggregation of
occupied receptors (Boulay and Paul, 1992). Since the stoichiometry of
the hIL5
receptor
chain interaction is 1:1, receptor
aggregation leading to signal transduction presumably must involve
factors other than IL5 and
chain alone. An
-
-IL5
heterotrimerization could provide the aggregation needed to trigger
signal transduction.
Affinity of Soluble and Membrane-associated Receptor
The affinity of IL5 for receptor was measured with
both shIL5R Subunit Are Similar But Lower Than That for
Heterocomplex
and shIL5R
-Fc by titration calorimetry and
real-time biosensor kinetic analysis. In both instances, the
K
values obtained were in the range of
2-8 n
M at 25 °C. We also observed that the IL5
binding affinity of the soluble form of the
receptor is very
similar to that of the membrane-bound, full-length form. Of note, the
2-8 n
M affinities reported here for soluble and
membrane-associated Drosophila-expressed receptor
chain
are somewhat lower than the 0.6-1 n
M values reported
before for
chain expressed in mammalian cells (Tavernier et
al., 1991, 1992; Takaki et al., 1993). The reason for
this difference is not fully understood at present.
chain forms strongly suggests the absence of
structure-energy coupling between IL5 binding and membrane-induced
structural constraints in the
receptor. This argues against the
possibility of transmembrane signal transduction occurring through the
receptor alone, in agreement with the cell proliferation studies
of Takaki et al. (1993).
,
is of quite high affinity. Nonetheless, the IL5 affinity of cells
expressing both
and
chains of receptor has been reported to
be greater than for the
receptor alone by a factor of 2 to 4
(Tavernier et al., 1991, 1992; Takaki et al., 1993).
Although this difference is rather modest, an increase in IL5 affinity
for
versus
alone is in agreement with the
binding energetics expected for a ligand-induced oligomerization
equilibrium ( cf., Wyman and Gill, 1990). The ability of IL5 to
induce
-
heterodimerization requires its affinity for the
complex to be higher than that for either
or
alone. Of note, while the increase is modest in hIL5 affinity for cells
expressing both
and
chains versus
chain
alone, the intrinsic affinity increase for the
heterocomplex
may be considerably higher but offset by free energy needed to drive
the receptor heterodimerization equilibrium. IL5-induced receptor
oligomerization is a part of the predicted model of IL5-receptor
interaction put forth below.
Binding Thermodynamics Suggest Receptor Isomerization in
the hIL5-shIL5R
The interaction analyses
carried out in this work suggest that conformational change occurs upon
IL5 Interaction
receptor
chain interaction. This conclusion was drawn
mainly from titration calorimetry data based on analysis of the binding
thermodynamics, in particular the binding entropy change (see below).
Biosensor-based kinetic analysis provided data consistent with this
conformational isomerization model.
chain can
be estimated from the thermodynamics of the interaction. Burial of
apolar residues in particular has long been known to be proportional to
the heat capacity change of a given protein-protein interaction
( cf., Tanford, 1980; Privalov and Gill, 1988). Recently,
Murphy and Freire (1992) have extended this approach by accounting for
the change in accessible surface area (
ASA) of both
apolar and polar residues from the experimental binding
C
according to the empirical
relationship,
for the total amount of surface area buried during
the binding of IL5 to the shIL5R
implies either a very large
interfacial surface between the two proteins or structure changes
coupled to the binding reaction. Previous work suggests that much of
the structural determinants for IL5-receptor binding lie in the A and D
helices of IL5 (Kodoma et al., 1991; McKenzie et al.,
1991; Shanafelt et al., 1991), and a model proposing the
involvement of the A and D helices has recently been put forward
(Goodall et al., 1993). The size of the accessible surface
area of a single A and D helix pair, as judged by visual inspection of
the crystal structure, is approximately 500 Å
, while
an entire face of the IL5 dimer exposes approximately 1900
Å
. Hence, either an entire face of the IL5 dimer is
involved in receptor binding, or conformational changes are coupled to
binding which cause burial of surface area in other regions of IL5
and/or its receptor. From mutagenesis studies,
(
)
at least significant numbers of residues on the hIL5 AD
surface can be changed to Ala without loss of shIL5R
binding. This
argues against binding through the entire face of the IL5 dimer and,
rather, for conformational change. Since IL5 is comprised of two
4-helix bundles, with helix sharing between the bundles, and shows
considerable thermal stability relative to shIL5R
,
(
)
significant conformational changes seem more likely to
occur in the receptor than in IL5 (although some degree on
conformational isomerization in IL5 itself cannot at present be
excluded). Cytokine receptor isomerization has been proposed recently
for leukemia inhibitory factor, and multiple kinetic dissociation rates
of leukemia inhibitory factor from its receptor as observed in a
cell-binding filter assay were proposed to reflect this isomerization
(Layton et al., 1994). For the IL5-receptor system, allosteric
modification of IL5 upon
chain binding has been hypothesized as a
possible way to explain both the lack of binding to a second
chain and increased affinity for the
chain (Devos et
al., 1993). Such IL5 allosterism, if it occurs, could involve
subtle conformational change and might occur in addition to the
conformational change of receptor proposed in the present study to
occur upon ligand binding.
binding
reaction is provided from their binding entropy changes. The total
entropy change (
S
) for binding has
contributions from several terms (Spolar and Record, 1994):
shIL5R
-Fc interaction (and also previously for the
interaction of hIL5 with shIL5R
(Morton et al., 1994))
are consistent with conformational isomerization such as that concluded
from titration calorimetry. Interestingly, in a previous biosensor
study (Fisher et al., 1994), the kinetics of binding of
oncoproteins to DNA also demonstrated multiphasic kinetics, and a
similar conclusion of conformational isomerization was proposed. It
must be cautioned however that, while multiple on and off rates suggest a more complex binding model than as shown in
Equation 4, the deviation from this simple model may be caused by
factors other than conformational isomerization, for example those that
derive from the biosensor method itself. These include immobilized or
soluble analyte heterogeneity, rebinding after dissociation, and
rate-limiting mass transport. In general, independent evidence is
required to test the hypothesis of receptor isomerization when this is
suggested by biosensor data. The results obtained in the current study
by titration calorimetry provide a step in that direction. Interaction
studies with mutant forms of hIL5 and receptor also would be of help.
Implications for the Mechanism of IL5 Receptor
Interaction and Signal Transduction
The data in the current
work, combined with the prevailing state of understanding on the
interaction of IL5 and its receptors ( cf. Kioke and Takatsu,
1994; Goodall et al., 1993), lead to a prediction of
IL5receptor interaction and signal transduction shown in Fig. 10.
Fig. 10 A depicts schematically both the 1:1 stoichiometry of
hIL5
chain interaction and the conformational isomerization
which have been observed in this study. Fig. 10 B represents a prediction of how conformational change, in
conjunction with binding of
chain, could lead to signal
transduction. This model accounts for the higher affinity of hIL5 for
,
heterocomplex versus
subunit alone
(Tavernier et al., 1991, 1992; Takaki et al., 1993).
It also accounts for evidence showing that IL5 can be cross-linked with
either
or
(Migita et al., 1991; Devos et
al., 1991). That IL5 can bind directly to the receptor
chain
fits with the view that, while the IL5 does not bind to
chain
alone, it can do so upon binding to the
chain (Shanefelt et
al., 1991; Lopez et al., 1992). The model in
Fig. 10B shows an absence of a tightly preformed
heterocomplex, a feature which also can be argued from the
previously observed cross-competition among IL5 and GM-CSF on cells
coexpressing the common
chain and the
chains specific for
the two cytokines (Tavernier et al., 1991). The presence of
preformed but weakly associated
heterocomplexes cannot be
ruled out by the current data. Fig. 10 B indicates that
IL5 may either bridge the
and
receptors or may induce a
conformational change in the
receptor which then allows for
direct interaction between the two receptor molecules. Both are
possible given the current data.
Figure 10:
Models of 1:1 complex of IL5 with
receptor chain and the conformational isomerization of IL5
receptor that may promote receptor heterodimerization with the
chain and consequent signal transduction. A, model showing 1:1
stoichiometry and conformational isomerization in hIL5-
chain
interaction. This model is drawn from the experimental data of this
study. B, an extension of model A, based on data in
this study and prevailing published data, depicting how the
and
chains may interact with each other upon association with hIL5.
Receptor subunit association is viewed as a possible trigger for signal
transduction. This view is analogous to the association of human growth
hormone receptor subunits upon interaction with human growth hormone
(Wells and de Vos, 1993).
The model as shown in
Fig. 10B for IL5 receptor activation resembles that of
growth hormone, in which signal transduction is implicated to require
hormone-induced receptor homodimerization (Wells and de Vos, 1993). On
the other hand, the model contrasts with that of the closely related
system GM-CSF, where pre-existence of a receptor heterodimer
has been postulated (Ronco et al., 1994). However, in that
work, the data were indirect, and at any rate the preformed complex
postulated could well be weak, at least in the absence of GM-CSF.
Finally, it should be noted further that, while the model in
Fig. 10B suggests formation of a 1:1:1 complex of hIL5
with
and
chains, greater states of aggregation, as
suggested by Boulay and Paul (1992), cannot be ruled out in particular
as they may occur on the cell surface. This possibility is suggested by
the observed tendency of both shIL5R
and shIL5R
-Fc to
aggregate in solution (Fig. 2). The potential for
chain to
dimerize in the absence of ligand (or
chain) has been
hypothesized recently (D'Andreas et al., 1994).
Figure 2:
Equilibrium sedimentation of hIL-5 and
shIL5R. A, global analysis of equilibrium sedimentation
data for IL-5. The upper three subpanels are the distributions
of residuals (observed-fitted values) for the analyses of the data in
terms of Equation 1 (see ``Experimental Procedures'') at
30,000, 20,000, and 25,000 revolutions/min, respectively. The lower
panel is the primary data taken at the same rotor speeds. Data
were taken 23, 73, and 91 h after the start of the run. The
concentration range examined in this analysis was from 10
to 10
M IL-5. The initial loading
concentration was
2
10
M ( A
=
0.4). B, global
analysis of equilibrium sedimentation data for shIL5R
. The
upper three subpanels are the distributions of residuals for
the analyses of the data in terms of Equation 1 (see
``Experimental Procedures'') at 15,000, 20,000, and 25,000
revolutions/min, respectively. The lower panel contains the
primary data taken at the same rotor speeds. Data were taken 22, 44,
and 66 h after the start of the run. The concentration range examined
in this analysis was from 10
to 10
M shIL5R
. The initial loading concentration was
6
10
M ( A
=
0.4). The solvent was 10 m
M sodium
phosphate buffer, 150 m
M NaCl, pH
7.4.
Final Comments
The Drosophila expression
system established in this study has enabled quantitative questions to
be addressed concerning the interaction of hIL5 and its receptor, by
providing both large scale production of soluble recombinant proteins
as well as membrane-incorporated full-length recombinant receptor. The
functional activity and, for hIL5, crystal structure confirm that the
Drosophila-expressed proteins are adequate surrogates of the
naturally occurring proteins for interaction analysis. The data show
the formation of a 1:1 hIL5shIL5R
complex of n
M affinity, which poses the possibility that one receptor molecule
can bind to both 4-helix bundle units of the hIL5 dimer simultaneously.
(The same may be true for the
chain.) The data also show that
essentially all of the binding energy of interaction of full-length
hIL5R
derives from its soluble domain. Finally, analysis of the
binding thermodynamics and kinetics suggests that conformational change
is likely to accompany hIL5
receptor interaction. Such
conformational isomerization could be an important component in signal
transduction. Beyond this study, the Drosophila expression
system promises to enable further mechanistic studies at both the
molecular and cell level, including high resolution structure
determination of hIL5
shIL5R
complex, effects of mutagenesis
on IL5-receptor recognition, and affinity measurements of the
chain when expressed in membranes.
Table:
Binding thermodynamics of human IL5 and its
soluble -receptor at 25 °C, pH 7.4, 20 m
M potassium
phosphate, 150 m
M NaCl
, soluble human interleukin 5 receptor
chain; shIL5R
-Fc, soluble human interleukin 5 receptor
-Fc chimera; tPA, tissue plasminogen activator.
as 67 and 44
°C, respectively, by monitoring changes in circular dichroism.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.