(Received for publication, November 5, 1996, and in revised form, January 13, 1997)
From the Department of Biochemistry and
Biotechnology, Royal Institute of Technology, S-100 44 Stockholm,
Sweden and § Pharmacia & Upjohn AB, Preclinical Research,
S-112 87 Stockholm, Sweden
Details of the signal transduction mechanisms of
the tyrosine kinase family of growth factor receptors remain elusive.
In this work, we describe an extensive study of kinetic and
thermodynamic aspects of growth factor binding to a soluble
extracellular human insulin-like growth factor-I receptor
(sIGF-IR) variant. The extracellular receptor domains
were produced fused to an IgG-binding protein domain (Z) in transfected
human 293 cells as a correctly processed secreted -
-Z dimer. The
receptor was purified using IgG affinity chromatography, rendering a
pure and homogenous protein in yields from 1 to 5 mg/liter of
conditioned cell media. Biosensor technology (BIAcore) was applied to
measure the insulin-like growth factor-I (IGF-I), des(1-3)IGF-I,
insulin-like growth factor-II, and insulin ligand binding rate
constants to the immobilized IGF-IR-Z. The association
equilibrium constant, Ka, for the IGF-I interaction
is determined to 2.8 × 108
M
1 (25 °C). Microcalorimetric titrations
on IGF-I/IGF-IR-Z were performed at three different
temperatures (15, 25, and 37 °C) and in two different buffer systems
at 25 °C. From these measurements, equilibrium constants for the 1:1
(IGF-I:(
-
-Z)2) receptor complex in solution are
deduced to 0.96 × 108 M
1
(25 °C). The determined heat capacity change for the process is
large and negative,
0.51 kcal (K mol)
1. Further, the
entropy change (
S) at 25 °C is large and negative. Far- and near-UV circular dichroism measurements display significant changes over the entire wavelength range upon binding of IGF-I to
IGF-IR-Z. These data are all consistent with a significant
change in structure of the system upon IGF-I binding.
Cellular receptor structure and function relationships have become
the focus of an increasing amount of research, relevant for biological
understanding as well as for possible pharmaceutical applications. The
insulin-like growth factor-I receptor
(IGF-IR)1 is a transmembrane
glycoprotein and belongs to the receptor tyrosine kinase family that
includes, among others, the insulin (insR), epidermal
growth factor, and platelet-derived growth factor receptors (1).
Insulin-like growth factor-I (IGF-I) is a major regulator of both
cellular growth and metabolism (2). The components of the IGF hormone
signaling system are tightly regulated, both in tissue-specific and
developmentally specific manners. The IGF molecular system contains
three known peptide ligands (IGF-I, IGF-II, and insulin), three types
of cellular receptors (IGF-IR, insR, and
IGF-II/mannose 6-phosphate), and six known subtypes of circulating IGF-binding proteins (IGFBPs) (IGFBP-1 to IGFBP-6). The molecular components of the IGF signaling system have been the subject of several
recent review articles (3, 4). The mechanism by which insulin and IGF
molecules interact with their respective receptor to mediate signaling
is still not understood in great detail at the molecular level. The
ligand-induced receptor activation has been suggested to involve a
conformational switch in the quaternary structure upon ligand binding,
with movements of the extracellular parts and a congregation of the
cytoplasmic tyrosine kinase regions to enable activation (5, 6).
However, the complexity of IGF-I signaling remains rather elusive
almost 10 years after the molecular cloning of the human IGF-I receptor
gene (7).
IGF-IR is active as a preformed heterotetrameric receptor
containing two extracellular -domains with ligand binding sites and
two transmembrane-spanning
-domains, also harboring the cytoplasmic tyrosine kinase activity (Fig. 1A). The
chains are disulfide-linked in a
-
-
-
arrangement with
possibly three disulfide linkages between the
chains and one
disulfide bond between each
-
chain pair (8). The gene is
transcribed and translated as a single
-
polypeptide chain and
post-translationally processed and assembled into a mature receptor. It
has been shown for the homologous insulin receptor that receptor
maturation is achieved through proteolytic processing, oligosaccharide
attachment, and disulfidelinked homodimerization during the
transport from endoplasmic reticulum to Golgi and further to the cell
surface (9).
In this paper, the ligand binding properties of a purified extracellular portion of the IGF-IR have been characterized using BIAcoreTM biosensor analysis. From the BIAcore data, molecular association and disassociation rates as well as binding equilibrium constants were calculated. Binding properties of the soluble IGF-IR receptor variant were analyzed for the ligands IGF-I, des(1-3)-IGF-I, IGF-II, and insulin, respectively. Near- and far-UV circular dichroism measurements were performed to establish an estimation of possible secondary structure changes upon ligand association.
Microcalorimetric titration binding experiments of IGF-I to the
heterotetrameric IGF-IR-Z were performed at three different
temperatures, from which enthalpies, H0,
Gibbs free energies,
G0, entropies,
S0, and heat capacity,
Cp0, of the binding process could be
calculated. In this work, we have used the model proposed by Freire
et al. (10-13) to analyze microcalorimetric titration data
of the binding of IGF-I to the heterotetrameric IGF-IR-Z.
From this analysis, the change in hydration-accessible polar and
nonpolar surface areas were calculated, as well as the entropic contribution to changes in conformational degree of freedom upon binding.
This is to our knowledge the first combined study, using both BIAcore and titration calorimetry analyses of ligand binding, of a growth factor receptor from the insulin-like growth factor family of tyrosine kinase receptors.
Native IGF-I, des(1-3)IGF-I, and IGF-II were produced in Escherichia coli and purified as described previously (14, 15). Recombinant human insulin was purchased from Sigma.
Production Vector ConstructionA vector was constructed for
the production of IGF-IR including an affinity handle for
purification and analysis purposes. Human IGF-I receptor cDNA was
cloned using linked reverse transcriptase-polymerase chain reaction of
placental total RNA (Clontech) into a vector containing cytomegalovirus
promoter and enhancer regions, a single Z domain gene, a stop codon
followed by SV40 poly(A)-signal and 3-untranslated regions. A sequence
coding for a thrombin-sensitive cleavage linker, SGLVPRGSG, was
inserted between the receptor
-3
and Z-5
coding regions at amino
acid position 933 according to the numbering of Ullrich et
al. (7). The outline of the expression cassette of the resulting
plasmid, pKGE978, is shown in Fig. 2.
Transfection and Expression
Human primary kidney 293 cells were transfected for transient expression using the calcium phosphate method (16). Cells were incubated for 24 h in Dulbecco's modified Eagle's medium/McCoy's 5A 1:1 (Life Technologies, Inc.) plus 5% bovine calf serum (HyClone), upon which the medium was replaced by serum-free medium and cells were incubated for another 24 h. Stable transfected cell lines were established from 293 human kidney cells transfected with pKGE978 and co-transfected with plasmid pKGE800 carrying a neomycin resistance gene. Selection was performed using G418, Geneticin, (Sigma) at 1 mg/ml.
Receptor PurificationCell media harvested after 48-72 h
of growth were centrifuged to remove cell debris and further diluted
with 1/2 volume of water and 1/10 volume of 10 × TST (1 × TST: 50 mM Tris-HCl, 150 mM
NaCl, 0.05% Tween 20, pH 7.4) to buffer the media to a pH suitable for the chromatography step. Diluted and buffered medium was filtered through a 0.22-µm filter to remove insoluble material prior to chromatography. Affinity chromatography purification was performed using an IgG-Sepharose FF column (Pharmacia Biotech, Sweden), previously equilibrated with 1 × TST. Unspecifically bound
proteins and impurities were removed with 10 column volumes of 1 × TST and 2 volumes of 5 mM ammonium acetate, pH 5.0. Bound fusion protein was subsequently eluted by 0.2 M
acetic acid, pH 2.8. Fractions were immediately neutralized through
collection in tubes containing 1/3 fraction volume of 1 M Tris-HCl, pH 8.0. The buffer of the eluted material was
exchanged to 10 mM HEPES, pH 7.4, through several rounds of
dilution and concentration using a Filtron ultrafiltration unit with a
30-kDa molecular mass cut-off (Filtron). Purified and concentrated
receptor was stored in aliquots at 80 °C.
Quantitative amino acid analysis was performed by acid hydrolysis of the peptide chain in 6 M HCl at 155 °C for 45 min, followed by analysis using an ion exchange column and ninhydrin derivative detection. The analysis was performed on a Beckman 6300 amino acid analyzer, equipped with a System Gold data handling system (Beckman).
The N-terminal protein sequence was determined using a HP G1005A protein sequencing system with on-line phenylthiohydantoin analysis, version 2.2 chemistry (Hewlett-Packard).
The oligosaccharide content of the receptor was determined by analysis of hexose content as described by Kenne and Strömberg (17). Briefly, hexose content was determined by acid hydrolysis and alditol acetate derivation of released sugars. Sugar alditol acetates were separated by gas chromatography-mass spectrometry on a fused silica capillary column (25 m × 0.2 mm) at 250 °C. An HP 5970B mass selective detector (Hewlett-Packard) was used for monitoring eluted peaks. Samples were compared with calibration curves constructed using defined sugar standards. N-Acetylneuraminic acid (sialic acid) content was analyzed by cleavage of the glycosylating carbohydrate chains with hydrogen chloride in methanol, transferring the sialic acid to its methyl ester methyl glycoside and further separation by gas chromatography.2 Protein homogeneity was evaluated by SDS-polyacrylamide gel electrophoresis (18) using NOVEX precast 4-20% gradient polyacrylamide gels (Novel Experimental Technology) stained with Coomassie Blue staining. The Z affinity tail was detected using semidry transfer to nitrocellulose membranes and peroxidase anti-peroxidase staining as detailed previously (19). Apparent molecular mass of both free and complexed receptor was determined using laser light-scattering analysis, performed with a Protein Solutions Dyna Pro 801 TC (Protein Solutions). The receptor was analyzed at a concentration of 1 mg/ml in 10 mM phosphate buffer, pH 7.4, at seven different temperatures in the range 4-25 °C.
Partial Reduction and Alkylation of the ReceptorPartial
reduction of receptor heterotetramers from (-
-Z)2 to
-
-Z was performed essentially according to the protocol of Sweet
et al. (20, 21). A 5-ml volume of 1 M Tris, pH
10.5, was added to 30 µg of receptor in 50 µl of 50 mM
Hepes buffer, pH 7.6, giving a final pH of 8.5. The mixture was
incubated at room temperature for 25 min before the addition of reduced
dithiothreitol to a final concentration of 2 mM. Following
a 15-min incubation, free thiols of the partially reduced receptor were
alkylated by adding iodoacetic acid to a final concentration of 100 mM. The alkylation reaction was allowed to proceed for 60 min at room temperature in the dark. The sample was then desalted on a
PD-10 Sephadex G50 column (Pharmacia Biotech, Inc.), preequilibrated with 50 mM ammonium acetate, pH 6.8, and lyophilized. The
partial reduction was analyzed using SDS-polyacrylamide gel
electrophoresis on a 4-20% precast NOVEX gradient gel stained using
Coomassie Brilliant Blue.
Analytical ligand binding assays were
performed using phosphate, pH 7.5, 150 mM NaCl, 0.05%
Tween 20 as assay buffer and 125I-labeled IGF-I (Amersham,
UK). Pre-IgG-coated Scintistrip microwell plates (Wallac, Finland) were
incubated with the receptor preparation. Plates were washed three times
with PBS-T and three times with assay buffer. One series of dilutions
was incubated with 125I-IGF-I (20,000-30,000 cpm/0.1 ml).
A second series was incubated with 125I-IGF-I and unlabeled
recombinant IGF-I (1 µM) for control of unspecific
binding. After incubation at 4 °C for 3 h or at room temperature for 1 h, plates were rapidly washed with assay buffer and allowed to dry. For competitive ligand binding assays, plates were
coated with a defined receptor concentration and washed. Receptors were
incubated with 100 µl of 125I-IGF-I in all wells. Second,
serial dilutions were made starting with 50 µl of
125I-IGF-I solution containing 3 µM unlabeled
IGF-I. After incubation, wells were treated as described above. Bound
radioactivity was measured using a counter (Wallac).
Far-UV circular dichroism spectra were recorded using a Jasco J 720 spectropolarimeter. Spectra were recorded from 250-184 nm at a step resolution of 0.1 nm using a scanning speed of 10 nm/min. Each shown spectrum is the average of three accumulated scans. Protein was dissolved in 15 mM potassium phosphate buffer, pH 7.0, to a concentration of 0.2 mg/ml. Cuvette path length was 0.1 cm. Actual protein concentration was determined using quantitative amino acid composition analysis. Secondary structure contents were estimated using the VARSLC1 variable selection software (22). The CD spectrum of the receptor was compared with a CD library containing reference spectra of 33 proteins. Two proteins were excluded in each iteration, creating 528 possible combinations of the first fit. The two least comparable proteins were removed from the reference set, and the iterative fitting was repeated until the set structure content criteria, more than 96% total secondary structure classified, and the root meant square difference to the reference set <0.3, were satisfied. Near-UV circular dichroism spectra were recorded from 320 to 240 nm using 0.5-cm cuvettes and a scan rate of 20 nm/min. The protein concentration was 0.3 mg/ml. Spectra shown are the average of five accumulated scans.
Biosensor AnalysisThe BIAcoreTM, Sensorchip
CM5, certified grade, Surfactant P20, and Amine coupling reagents,
N-ethyl-N
-(dimethylaminopropyl)-carbodiimide, N-hydroxysuccinimide, and ethanolamine
hydrochloride were obtained from Pharmacia Biosensor (Sweden).
All other buffer chemicals were obtained from
Sigma or Fluka (Switzerland). Measurements were
performed with IGF-IR-Z as the immobilized acceptor
molecule. The receptor was immobilized via primary amine groups as
described previously (23) utilizing N
-ethyl-N
-(dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide coupling reagents. Protein absorption and coupling was performed at pH
4. Immobilizaton was performed at 5 µl/min using 1 × HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.05% P20) as the driving buffer. The final level
of IGF-IR-Z immobilization was between 6500 and 7500 resonance units. All kinetic experiments were performed using
1 × HBS as the driving buffer at a flow rate of 8 µl/min.
The injection of analyte was controlled using the "kinject" command
in the BIAlogueTM control software. Ligand sample was
injected twice at six different concentrations in random order over the
same surface in each series of measurements. Immobilized
IGF-IR-Z was regenerated after each cycle by injection of
12 µl of acidic regeneration solution containing 0.3 M
sodium citrate, pH 5, and 0.4 M NaCl. Kinetic measurements
were performed by injection of each analyte for 300 s followed by
disassociation in buffer flow for 400 s. The temperature in all
kinetic experiments was 25 °C.
The kinetic parameters were calculated using the kinetics evaluation software package, BIAevaluation 2, (Pharmacia Biosensor). The theory of BIAcore measurement techniques and calculations has been extensively described (for a review, see Ref. 24).
Titration MicrocalorimetryThe titration microcalorimetry
experiments were performed at 15, 25, and 37 °C using a 2-ml
titration microcalorimetric stainless steel vessel for the multiple
channel microcalorimetric system TAM (Thermometric AB, Sweden) (25,
26). Another 2-ml vessel, lacking stirring facilities and containing
0.8 ml of water, was used as a calorimetric reference in the twin
microcalorimetric unit. The noise level was estimated to be 10
nanowatts. Electrical calibration was performed in connection with each
experiment, with regard to both the energy and the time constants of
the instrument. The calorimetric vessel was loaded with 900 µl of 3 µM IGF-IR-Z soluble receptor. At each
titration, 25-30 aliquots of 4 µl of 65 µM IGF-I were
added with a 6-7-min interval between each injection (27) using a
Hamilton syringe attached with a hypodermic needle mounted on an
automated motor-driven pump. To correct for dilution enthalpies,
additional dilution experiments where IGF-I was added in the buffer
solution alone were performed. The IGF-IR-Z solutions were
prepared by exhaustive dialysis against PBS (20 mM sodium
phosphate, 145 mM NaCl, pH 7.40) and HBS (20 mM HEPES, 145 mM NaCl, pH 7.40), respectively. The IGF-I
solutions were prepared by dissolving freeze-dried desalted stock
solution in the same buffer solution that the IGF-IR-Z
solutions had been prepared in. The PBS buffer system was used in the
experiments at 15, 25, and 37 °C, while the HBS buffer system was
used in additional experiments at 25 °C. The concentrations of the
proteins were obtained from amino acid analysis.
The equilibrium constant was estimated by nonlinear regression according to Marquardt-Levenberg (28), where the enthalpy was kept constant at a value arrived at from an individual enthalpy determination of the binding process. In this way it is possible to minimize the number of unknown parameters and neglect any fitting correlation of the fitted parameters.
Thermodynamic AnalysisThe binding of a ligand to a protein
and protein folding are thermodynamically analogous processes. Except
for hydrogen bonding, electrostatic interactions, or other specific
interactions, the most important contribution to the thermodynamic
properties is the change in hydrophobic hydration. Changes in hydration
can be a result of changes in hydration of the binding surfaces of both
protein and ligand as well as changes in hydration due to conformational changes of the protein. The heat capacity change, Cp0
(
Cp0 = d
H0/dT), is the
thermodynamic property that is most effected by changes in hydrophobic
hydration. When transferring a nonpolar surface from a nonpolar
environment to an aqueous environment, e.g. dissociation of
protein-ligand complexes or unfolding of proteins, the
Cp0 is large and positive. A striking
feature of transferring a small hydrocarbon compound from a nonpolar
environment to an aqueous solution is the high degree of group
additivity to the heat capacity (29-31). The group additivity rules
can be translated into models where the thermodynamic properties are
functions of accessible solvent surface area,
ASA.
Sturtevant (32), Livingstone et al. (33), and Freire
et al. (11) have similar approaches to divide the
contributions from hydration/dehydration of nonpolar and
polar-accessible surface areas,
ASAnp and
ASApol, respectively, by parameterizing
H0 and
Cp0.
It has been shown that the major contributions to
Cp0 and
H0
for protein folding/unfolding or protein-ligand interactions can be
rationalized in terms of
ASA (11, 32, 33).
From the temperature dependence of the enthalpy, the heat capacity was calculated.
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
The intrinsic binding enthalpy, Hint, and the
number of linked protons,
n, is calculated from Equations
4 and 5.
![]() |
(Eq. 4) |
![]() |
(Eq. 5) |
When analyzing the calorimetric data we have used the method developed
by Freire et al. (11) and the parameters from Xie and Freire
(13) for calculating changes in hydration-accessible nonpolar surface
area, ASAnp, and accessible polar surface
area,
ASAp, upon binding. When corrections
have been made for proton linkage, as described by Equations 4 and 5,
the enthalpy of binding can be parameterized as follows,
![]() |
(Eq. 6) |
![]() |
(Eq. 7) |
The total change in entropy, S0, upon binding
can be divided into a sum of different contributing effects.
(i) The contribution from the change in hydration upon binding,
Shyd, at temperature T is
calculated from the heat capacity change using a reference temperature,
Ts;
![]() |
(Eq. 8) |
(ii) There are contributions to the total entropy change due to
reductions in rotational/translational degrees of freedom, Srot/trans. Kauzman (36) and Murphy et
al. (37) have shown that
Srot/trans is
well approximated by the cratic entropy,
8 cal K
1
mol
1, for 1:1 binding stoichiometry.
(iii) The entropic contribution due to the change in the number of
particles in the system, Sno part, is a
statistical entropic effect.
![]() |
(Eq. 9) |
In the initial state for a 1:1 stoichiometry there are two
particles, while in the final state there is only one
particle. R is the gas constant (R = 1.987 cal (mol K)1. Thus,
Sno part is for 1:1
binding Rln(1/2) =
1.1 cal K
1
mol
1.
(iv) In the protein-protein interactions there can be changes in
ionization of amino acid residues. This will contribute to the total
entropy change as an ionization entropy contribution, Sion. The magnitude to this contribution is
dependent on which type of residue that is involved in the change in
ionization.
(v) In the binding process involving a protein, it is likely that there
will be changes in conformational degrees of freedom. This can be due
to conformational changes in the peptide backbone and/or conformational
changes of individual residues. The changes in conformational freedom
will contribute to the total changes in entropy,
Sconf.
Adding up the contributions (i-v) we arrive at the following expression for the total change in entropy upon binding.
![]() |
(Eq. 10) |
The extracellular portion of the receptor was produced as a soluble secreted molecule with an affinity handle fused to the C-terminal end. The use of the Z affinity tail has been successfully implemented in the production of a large variety of proteins in various prokaryotic production systems (38) and is in this report used in a eucaryotic host system.
Cloning/SequencingThe receptor was cloned and sequenced in the production vector pKGE 978, and the sequence was found to be fully in agreement with the published sequence (7).
Cell GrowthHuman primary kidney 293 cells were transfected for transient expression of the IGF-I receptor. In addition, 293 clones expressing the receptor were selected for establishment of stable cell lines. The selected 293 cell line yielded high and stable expression combined with a minimum of proteolytic degradation.
Protein CharacterizationThe small scale cell growth used in
the initial experiments, up to 50-ml flasks, gave a production yield
ranging from 1 to 5 mg of IgG-purified sIGF-IR-Z/liter of
conditioned media. The purity of the receptor was determined using
SDS-polyacrylamide gel electrophoresis (Fig. 3A). Detection of the Z affinity handle using
peroxidase anti-peroxidase staining (Fig. 3B) reveals a
single band in the unreduced sample (lane B2) corresponding
in size to full-length soluble receptor. In the reduced sample
(lane B1), a band corresponding to the size of the
-Z-chain is stained. No additional bands, and therefore no
detectable degradation products, containing the Z tail were found. The
determined amino acid composition corresponds to what is expected from
theoretical values (data not shown). Five cycles of N-terminal
sequencing revealed two different sequences corresponding to a
correctly processed
-chain N terminus after signal peptide removal
and a postcleavage
-chain N terminus, respectively.
Receptor processing and association status were analyzed by partial
reduction analysis. This analysis is based on the fact that the -
interchain (class I) disulfides are more susceptible to reduction than
intrachain (class II) disulfides (20). Limited reduction results in the
appearance of
-
-Z in addition to the parent
(
-
-Z)2 receptor. The gel in Fig. 3A shows
the presence of one species in the unreduced sample (lane
A2), corresponding approximately to the size of
(
-
-Z)2 receptor. The totally reduced sample
(lane A1) exhibits two bands of approximately 134 and 70 kDa, corresponding to
- and
-Z chains, respectively. The
partially reduced sample (lane A3) shows the presence of two
bands corresponding to nonreduced receptor and a band of intermediate
molecular mass, 180 kDa, which may correspond to the
-
-Z half
receptor. The molecular masses determined may deviate from theoretical
values due to changes in gel mobility originating from glycosylation and different levels of intrachain reduction in the samples as well as
possible charge differences originating from the alkylating agent.
Glycosylation analysis of the receptor reveals a sugar content of
26.1% by mass. The composition of associated sugars is as follows:
fucose, 2.2.%; mannose, 7.7%; galactose, 4.6%; and glucosamine, 9.6%. The sialic acid component was found to be 2%. Total molecular mass based on the theoretical molecular mass, derived from amino acid
composition plus sugar content, is 280 kDa for the
(-
-Z)2 receptor. Attempts to determine the molecular
mass of the complete receptor assembly using mass spectrometry did not
succeed using either laser desorption or electrospray methodology.
Laser light-scattering analysis of both free and ligand-complexed receptor indicates an apparent hydrodynamic radius slightly larger than expected from theoretical calculations. The calculated molecular mass of free receptor at 25 °C was approximately 370 kDa, and the mass of the complex was 410 kDa. The determined value may differ up to 20% from the actual value in this analysis. From the light scattering data the receptor was found to be monodisperse, indicating a homogenous receptor population. The difference in size between free and complexed receptor is much larger than the actual size of the ligand (7.5 kDa) and may reflect different deviations from the theoretical globular shape assumed in the calculation of molecular mass.
Circular DichroismCD spectroscopy was used to detect
possible changes in secondary structure content upon ligand binding.
The far-UV spectra of free and ligated receptor are shown in Fig.
4A. The spectrum of the free IGF-I ligand is
subtracted from the complexed receptor spectrum. Calculations of the
secondary structure components using VARSLC1 variable selection suggest
a structure content for the free receptor of 13% -helical, 26%
anti-parallel
-sheet, 9% parallel
-sheet, 18% turn, and 35%
unstructured. Spectra of IGF-IR-Z complexed with an excess
of ligand (IGF-I) (spectra of the ligand subtracted) reveal small but
reproducible differences over the wavelength range suggesting that
these differences observed are a direct consequence of binding IGF-I to
its receptor. There is a decrease in negative ellipticity for the
complex over the entire range. The calculated structural element
content of complexed receptor is 14%
-helical, 28% antiparallel
-sheet, 7% parallel
-sheet, 20% turn, and 31% unstructured.
The absolute values of secondary structure may contain added
uncertainties due to contribution to negative ellipticity from
associated oligosaccharides below 210 nm.
The near-UV spectra of the free receptor, of the ligand-complexed receptor with ligand signal subtracted, and of the sum signal of free receptor and ligand, are shown in Fig. 4B. In contrast to the far-UV spectra, there is an increase in negative ellipticity upon ligand binding in the near-UV spectra. The negative ellipticity at 217 nm is decreased by 9%, and at 286 nm it is increased by 57%. The near-UV spectra is sensitive to aromatic side chain positions and cysteine bridge conformations.
Ligand Binding AssayThe binding displacement of radiolabeled
IGF-I to purified IGF-IR-Z, using IGF-I as a tracer, is
shown in Fig. 5. The half-maximal inhibiting
concentration (IC50) value calculated from the binding curve is 0.5 nM.
Biosensor Analysis
BIAcore kinetic measurements were
performed for a series of known ligands to the IGF-IR:
IGF-I, IGF-II, des(1-3)-IGF-I, and insulin. IGF-IR-Z was
immobilized, and sensorgrams for the different ligand interactions are
shown in Fig. 6. A summary of the calculated rate and
equilibrium constants is found in Table I. IGF-I
affinity was determined in three separate runs using duplicate
injections of each concentration, and affinity for the other ligands
was determined in one run, also using duplicate injections. The
cumulative error in the determined association constants from all runs
was estimated to be less than 12%, calculated as the square root of
the sum of the squares of the errors in amino acid analysis, pipetting,
and data fitting. Determined kinetic parameters (Table I) indicate that
differences in both association rates and disassociation rates underlie
the observed differences in calculated association equilibrium
constants for the tested ligands. The des(1-3)-IGF-I variant has an
association rate that is about 1.5 times faster than that of native
IGF-I, while the association rate for IGF-II is about 2.8-fold slower than IGF-I. IGF-I and des(1-3)-IGF-I have similar disassociation rates, while that of IGF-II is 2 times faster than that of IGF-I. The
insulin association was too slow to be determined, using BIAcore technology at the conditions used. Ligand association was analyzed for
insulin at concentrations up to 500 times higher (0.17 mM) than those used for IGF-I. The differences in disassociation rate between the ligands are less pronounced, insulin excluded, than the
association rates. The data for insulin disassociation is best fitted
to a two-site model in contrast to the other ligands. Out of the two
distinct disassociation rates, one is 100 times faster than that of
IGF-I, 180 (s1·10
3) and the other is 10 times faster, 18 (s
1·10
3). The calculated
association equilibrium constants for IGF-IR-Z are thus
about 2-fold higher for des(1-3)-IGF-I than for native IGF-I, while
the value for IGF-II is about 5-fold less, accounted for both by slower
association and faster release from the receptor.
|
The measured heats of binding were at all
temperatures exothermic. The largest heat was obtained at the first
injection at each titration series and ranged from 30 to 65 µJ (Fig.
7). At 15 and 25 °C, the measured heat values in the
PBS buffer were below the limit of accurate determination of
equilibrium constants. However, due to the larger enthalpy at 37 °C
in PBS and at 25 °C in HBS, data could be fitted to a 1:1 binding
model with acceptable statistical output (Fig. 8). Other
thermodynamic binding models, e.g. models including a
complex with stoichiometry of 1:2 or 2:1 (IGF-1:IGF-IR-Z),
were also tested to establish the most probable thermodynamic model for
the binding process. These other models could be disregarded based on
statistical analysis. At 15 and 25 °C in PBS buffer, the enthalpies
of binding were calculated from total integral heats. The results from
the titration calorimetric experiments are summarized in Table
II. The results from the analysis of the thermodynamic
data are listed in Table III. The thermodynamic
properties are dominated by the large negative heat capacity. This is
typical for protein-protein interactions in which the hydration of
nonpolar residues is reduced. Due to the large heat capacity change,
the enthalpy and the entropy are strongly
temperature-dependent. As outlined under "Experimental
Procedures," the large negative heat capacity change can be explained
by dehydration of large hydrophobic surface areas. The large and
negative enthalpy also implies a strong temperature dependence of the
affinity constant, which indeed can be observed by the difference in
affinities at 25 and 37 °C. The number of linked protons in the
reaction was determined to be 3.1 at pH 7.4. Important contributions
to the entropy change upon binding comes from the solvent,
Shyd, stemming from the burial of hydrophobic
groups in the binding process and due to a decrease in the
conformational degree of freedom in the IGF-I and IGF-IR-Z
molecules. This contribution was calculated to be
131 cal K
1 mol
1 in total, at 25 °C. For the
IGF-I·sIGF-IR-Z system, there is no detailed
structural information at hand. Thus, it is not possible to account for
any specific ion interactions due to deprotonation of amino acids.
Additional structural information can to some extent revise the sizes
of the contributions to the different thermodynamic properties.
However, since the major contributions to enthalpy and heat capacity
stems from changes in hydration of the two proteins, the general
features of the contributions to the thermodynamic properties can be
judged to be valid within the experimental uncertainties.
|
|
According to the analysis of the thermodynamic properties, there is a
significant reduction in the degrees of freedom for the proteins upon
binding, which is manifested by the large and negative
Sconf (Table III). Apart from the binding
surface areas of IGF-I and IGF-IR-Z, the large change in
total accessible surface area can have its origin in changes in
hydration of the proteins due to conformational changes. The accessible
surface area for free IGF-I is 4070 Å2, based on the
NMR minimum average structure (39) and the Lee and Richards algorithm
(40) implemented in the program INSIGHT (Biosym).
In this paper, we have described the ligand binding properties of recombinant sIGF-IR. The detailed characterization of the IGF-IR ectodomain in terms of ligand binding kinetic properties, secondary structure analysis, and thermodynamic properties, with the finding of specific proton release and receptor conformational changes, as well as the 1:1 receptor:ligand stoichiometry, are important contributions to the understanding of the IGF-I molecule signaling mechanism and specificity.
The titration microcalorimetry-derived thermodynamic parameters, and in particular the large entropy loss upon ligand binding, are consistent with a conformational stabilization induced by ligand binding. Part of this stabilization could be induced by a conformational change. The titration microcalorimetry data presented here suggest that some 4700 Å2 get depleted of water when IGF-I binds to IGF-IRZ. This can be either through the burial of binding surfaces on both the ligand and the receptor or from structural changes leading to a reduction of solvent exposed surface in either of the molecules. Assuming rigid molecules, this would mean that more than half ((4675/2)/4070 = 0.57) of IGF-I gets buried upon binding. This is an unlikely large portion of the molecule, supporting the hypothesis that a conformational change does occur in the receptor. The CD analysis of the IGF-I receptor ectodomain suggests that the secondary structure of the receptor changes upon ligand binding. The far-UV CD spectra of free and IGF-I-ligated IGF-IR-Z show small changes in negative ellipticity (Fig. 4), and the near-UV spectral changes are somewhat larger, but in both ranges the changes are reproducible and significant. The difference in the spectra between the complex and the sum of the signals of free receptor and free ligand indicates that changes in position of aromatic side chains and, possibly, cysteine bridge dihedral angels occur in the receptor ectodomains upon ligand association. These changes could be due to secondary structural changes, quaternary structural changes, or both (41).
The three-dimensional structure of the insulin receptor cytoplasmic
tyrosine kinase domain has been determined using protein crystallography (6). These data suggest that the activation of the
tyrosine kinase involves intradomain transphosphorylation made possible
by induced close contact of the domains and structural rearrangements
in the protein kinase active site region. Thus, it seems possible that
signal transduction across the membrane is the result of a
ligand-induced quaternary structural change. The nature of this change
may, for instance, involve a scissors-like movement of the receptor
chains, a twist of the chains, or a combination of these movements.
Binding of ligand to both IGF-I and insulin receptors is proposed to
involve negative cooperativity (42). The mechanism has been suggested
to involve the presence of both high and low affinity binding sites on
each subunit. A ligand might bind to a high affinity site on one
subunit inducing conformational changes enabling the ligand to bridge
the two subunits by also binding to the low affinity site on the second
subunit. Our studies on IGF-IR-Z give a stoichiometry of
1:1 (IGF-I:(
-
-Z)2) with high affinity, which could
include two separate binding sites.
The relative potency of binding to the produced IGF-I receptor for the
different ligands is in accordance with previous investigations performed using competitive ligand methods (43). The des(1-3)-IGF-I exhibits a faster association rate than native IGF-I and thus a higher
affinity. IGF-II has 5 times lower affinity than IGF-I due to both
slower on rates and higher off rates. The observed discrepancies in
affinity constant between the BIAcore methodology and the titration
microcalorimetry are in accordance with previous studies and may be due
to higher degree of conformational freedom for the IGF-IR-Z
molecule in solution than when it is immobilized on a surface matrix.
This reduction in conformational degrees of freedom upon surface
immobilization would increase the apparent affinity for the ligand. The
receptor binding properties of a number of IGF-I mutants have been
tested using both BIAcore technology and classical radiolabeled
competition assays.3 Such studies show that
although the absolute value of binding may vary between used
methodologies, a series of molecules will have the same relative order
of affinities, disregarding the method used. Similar conclusions can be
made also for the interaction of human growth hormone mutants to the
human growth hormone receptor (44) and for binding of Z mutants and
their binding to IgG (45) using displacement and BIAcore methods. In
addition, the validity of association equilibrium constants determined
by BIAcore kinetic analysis has been addressed in works by Ladbury (51)
and Morton et al. (52); in both works the combination of
biosensor methodology and isothermal titration calorimetry was used to
characterize receptor-ligand interactions. Ladbury and co-workers (51)
found that only steady state measurements were reliable in the
biosensor setup used. In the work by Morton et al. (52),
good agreement was found between kinetic and steady state biosensor
measurements as well as titration calorimetry-derived values. We
believe the presented kinetic data to be correct based on the
following. The association is between a small monomeric ligand and a
large immobilized monodisperse receptor, thus minimizing the
possibilities of avidity effects, using self-associating proteins or
mass transport limitations, that occur when a large receptor associates
with a small immobilized ligand. The plot of ks
against concentration, used to determine the association rate
(kon), was linear in the used concentration range. The disassociation rate does not change with concentration in
the highest concentrations used, and the addition of free IGF-I-binding IGFBP-1 protein in the disassociation phase does not increase koff, demonstrating that detectable rebinding
does not occur. The obtained rate information provides the opportunity
to more fully quantitate the behavior of the ligands and the effects of introduced mutations in IGF-IR ligands. The association
rate of insulin binding to IGF-IR-Z is too low to be
determined using the BIAcore; it is possible, however, to calculate the
disassociation rate constant from the data. Interestingly, in contrast
to the other ligands, a two-site model gave the best fit for insulin
disassociation. The rates of disassociation are approximately 10 and
100 times faster, respectively, for the two insulin off-rates than the
corresponding rate for IGF-I. A hypothetical explanation for this
difference is that IGF-I binds to the IGF-I receptor and induces a
conformational change by simultaneously bridging the high and low
affinity sites (42). Insulin, on the other hand, binds to the receptor,
one molecule to each subunit, with significantly lower affinity but
is unable to induce conformational changes and is therefore rapidly
disassociated from these two sites. Analysis of ligand binding in the
presence of different antibodies directed toward the IGF-I receptor
(46) shows that signal transduction may be blocked independently of
ligand binding. This might be the case if the antibody would bind
outside the ligand binding site and block a conformational change, thus
inhibiting further signal transduction. In the study by Soos et
al. (46) there is a further observation that antibodies
potentiating IGF-I ligand binding also increase the affinity of insulin
for the IGF-I receptor up to 50-fold. These findings are consistent
with the thought that insulin on its own is unable to bridge between
the
-subunit ligand-binding regions of the IGF-I receptor. The
antibody could possibly switch the receptor conformation to an
"insulin-competent" state by locking the subunits in a conformation
allowing formation of an insulin high affinity receptor complex.
It has previously been shown by Gual et al. (47), using
conformational sensitive antibodies, that the full-length IGF-I receptor undergoes an autophosphorylation-induced conformational change
in its cytoplasmic region, which is distinctly separate from
ligand-induced conformational changes. In this work we have presented
data supporting a ligand-dependent conformational change in
the extracellular domains of a kinase-deleted IGF-I receptor. Two
electron microscopy studies of the structure of the highly homologous
insulin receptor have previously been published (48, 49). Christiansen
and co-workers (48) studied the structure of full-length receptor from
placental membrane preparations, both in the presence and absence of
insulin. They found two different forms of the receptor, one shaped
like an elongated T and a second more X-like in structure. The addition
of insulin did not yield a single conformation, but the possibility
could not be excluded that structural changes may accompany ligand
binding and receptor phosphorylation. Schaefer et al. (49)
studied the insulin receptor extracellular domains, using a construct
analogous to the extracellular IGF-IR presented in this
work and found forms of structures similar to those found by
Christiansen and collaborators. These studies support the theory of
large flexibility of the receptor
domains. The speculation that the
conformations found in these reports represent the structures of
receptor with or without ligand present is therefore appealing.
Further, Schaefer et al. (49) applied CD analysis to the
insulin receptor and concluded that changes in secondary structure
content occurred when insulin was added.
Detailed characterization of ligand binding of extracellular receptors requires a convenient way to produce sufficient amounts of the mature protein. We have demonstrated the successful use of an affinity handle fusion protein expression system for the production of large amounts of a soluble form of IGF-IR. Purification using the Z:IgG affinity system has high specificity and gives an essentially pure receptor preparation using a single-step chromatography procedure (Fig. 3). The IgG(Fc)-binding Z protein affinity handle, derived from the B domain of Staphylococcal protein A, has been extensively used in a large number of biotechnological applications for the production and purification of recombinant proteins (38). Growth impairments or toxicity effects have not been detected as a result of the production of this prokaryotic protein sequence in the 293 cell line used. The produced receptor variant displays limited, if any, proteolytic degradation even when harvested after prolonged cell growth.
The data presented in this paper support the hypothesis that extracellular IGF-I receptor domains undergo a conformational change upon ligand binding that includes changes in both the amount of accessible surface area and the amount of secondary structure content. The release of protons in the binding process may originate from formation of salt bridges between receptor and ligand. It has been shown by mutational analysis that the charged IGF-I C domain residues are important for high affinity IGF-I receptor binding (50). The proton release could also be part of the conformational change in the receptor structure. These two different possibilities cannot be distinguished without detailed structural or mutational analysis.
In this study, we have shown that the produced soluble IGF-IR-Z binds one IGF-I molecule with high affinity and is capable of structural changes, possibly those involved in the activation of signal transduction. The lack of transmembrane and cytoplasmic regions does not destroy high affinity ligand binding capacity.
Signhild Strömberg is acknowledged for performing the carbohydrate analysis, Carina Nordström for performing light-scattering analysis, Dr. Peter Lind for suggesting the concept of Z-fused receptor production, and Dr. Tomas Lundqvist for productive discussions concerning receptor structures and mechanisms.