From the The determinants of insulin-like growth factor
(IGF) binding to its binding proteins (IGFBPs) are poorly characterized
in terms of important residues in the IGFBP molecule. We have
previously used tyrosine iodination to implicate Tyr-60 in the
IGF-binding site of bovine IGFBP-2 (Hobba, G. D., Forbes, B. E., Parkinson, E. J., Francis, G. L., and Wallace, J. C. (1996) J. Biol. Chem. 271, 30529-30536). In this
report, we show that the mutagenic replacement of Tyr-60 with either
Ala or Phe reduced the affinity of bIGFBP-2 for IGF-I (4.0- and
8.4-fold, respectively) and for IGF-II (3.5- and 4.0-fold,
respectively). Although adjacent residues Val-59, Thr-61, Pro-62, and
Arg-63 are well conserved in IGFBP family members, Ala substitution for
these residues did not reduce the IGF affinity of bIGFBP-2. Kinetic
analysis of the bIGFBP-2 mutants on IGF biosensor chips in the BIAcore
instrument revealed that Tyr-60 The insulin-like growth factors
(IGF-I1 and IGF-II) are
polypeptides that play a central role in vertebrate growth and
development by stimulating cellular proliferation and differentiation
(recently reviewed in Ref. 1). The biological activities of the IGFs are mediated mainly through the type 1 IGF receptor which is found on
the surface of most cell types (1, 2). In turn, the bioavailability of
the IGFs is regulated by a family of IGF-specific binding proteins (IGFBPs). The IGFBP family consists of six high affinity IGFBPs (IGFBP-1 to 6) (reviewed in Refs. 3 and 4) and possibly an additional
four proteins that can associate with IGFs with lower affinity (5, 6).
Conserved gene structures and the high degree of sequence identity
between the high affinity IGFBPs suggest that these proteins possess
three domains and a common IGF binding motif (3, 4, 7). The sequences
of two of these putative domains, the N- and C-terminal cysteine-rich
domains, are highly conserved. Where the IGFBP family members differ is in the middle domain and in the possession of phosphorylation and
glycosylation sites or sites of association with other biomolecules such as heparin or the integrin receptor (3, 4).
At the molecular level, the IGFs have been well characterized. High
resolution NMR structures of both IGF-I (8) and IGF-II (9) have been
determined, and the overlapping regions that are responsible for IGFBP
and receptor interactions have been identified by chemical modification
(10, 11), epitope mapping (12), and by mutagenesis (13-18). However,
insight into the overall structure of the IGFBPs has been restricted to
multiple sequence alignment and secondary structure prediction (19).
Furthermore, the growing number of IGFBP mutagenic studies described to
date has focused on aspects of IGFBP biology such as heparin binding (20), integrin receptor binding (21), extracellular matrix binding
(22), specific proteolysis (23, 24) or phosphorylation (25) rather than
the systematic identification and characterization of a common IGF
binding motif.
In terms of the IGF-binding site, both the N- and C-terminal
cysteine-rich domains of IGFBPs are believed to participate. This is
suggested by the observations that N-terminal cysteine-rich domains of
IGFBP-1 (26), IGFBP-3 (27), IGFBP-4 (24), and IGFBP-5 (28) and
C-terminal cysteine-rich domains of IGFBP-2 (29, 30) and IGFBP-3 (31)
all possess residual IGF binding affinity. Yet, the specific residues
of IGFBPs that are directly involved in IGF binding have not been
identified.
The rationale for this study is based on our observation that Tyr-60
was protected from iodination in the IGF·bIGFBP-2 complex (32).
Furthermore, when Tyr-60 was iodinated, it caused a reduction in the
binding affinity of bIGFBP-2 for the IGF ligand. However, the residues
Val-59, Thr-61, Pro-62, and Arg-63 could also conceivably play a role
in IGF binding that is disrupted when Tyr-60 is iodinated. These latter
residues are highly conserved across the whole IGFBP family, whereas
all of the described IGFBP-1 sequences from various species possess an
alanyl rather than tyrosyl residue at the position corresponding to
Tyr-60 in bIGFBP-2, as shown in Fig. 1.
Therefore, in order to determine which residues in the Tyr-60 region of
bIGFBP-2 do influence IGF binding, alanine-scanning mutagenesis has
been performed across residues 59 and 63 inclusive. Tyr-60 has also been substituted with Phe to distinguish between the hydrogen bonding
properties and the hydrophobic and aromatic properties of the tyrosyl
side chain with respect to IGF binding.
Cooperative Research Centre for Tissue
Growth and Repair,
Pharmacia & Upjohn AB, Metabolic Diseases Research,
S112 87 Stockholm, Sweden
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Phe bIGFBP-2 bound to the IGF-I
surface 3.0-fold more slowly than bIGFBP-2 and was released 2.6-fold
more rapidly than bIGFBP-2. We therefore propose that the hydroxyl
group of Tyr-60 participates in a hydrogen bond that is important for
the initial complex formation with IGF-I and the stabilization of this
complex. In contrast, Tyr-60
Ala bIGFBP-2 associated with the IGF-I
surface 5.0-fold more rapidly than bIGFBP-2 but exhibited an 18.4-fold
more rapid release from this surface compared with bIGFBP-2. Thus both
the aromatic nature and the hydrogen bonding potential of the tyrosyl
side chain of Tyr-60 are important structural determinants of the
IGF-binding site of bIGFBP-2.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (18K):
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Fig. 1.
Sequence homology of the IGFBP family across
the "Tyr-60 region." The Tyr-60 region lies in the N-terminal
cysteine-rich region of the IGFBP family sequences and is found between
the 9th and 10th cysteine residues from the N terminus (corresponding
to Cys-57 and Cys-64 of bIGFBP-2, respectively). The sequences shown
are a summary of 27 aligned IGFBPs using the single letter amino acid
code, representing a range of species including bovine (b),
chicken (c), human (h), murine (m),
ovine (o), porcine (p), and rat (r).
All of the observed sequence variation in this region is shown. The
square open brackets ([) indicate species variation within
an IGFBP type, and complete sequence identity with the bIGFBP-2
sequence is denoted by a dash ( ). Percent identity relative to
bIGFBP-2, at each amino acid position is as follows: Cys-57 (100),
Gly-58 (100), Val-59 (96), Tyr-60 (85), Thr-61 (93), Pro-62 (56),
Arg-63 (82), and Cys-64 (100).
In this report, the IGF binding characteristics of Val-59 Ala,
Tyr-60
Ala, Tyr-60
Phe, Thr-61
Ala, Pro-62
Ala, and Arg-63
Ala bIGFBP-2 have been compared with bIGFBP-2 by Western ligand blot, solution binding assays, and in BIAcore experiments with
immobilized IGFs. Mutagenic replacement of Tyr-60 with either Ala or
Phe reduced the affinity of bIGFBP-2 for IGF-I (4.0- and 8.4-fold,
respectively) and for IGF-II (3.5-fold and 4.0-fold respectively). In
contrast, Ala substitution for adjacent residues Val-59, Thr-61,
Pro-62, and Arg-63 did not greatly affect the IGF affinity of
bIGFBP-2.
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EXPERIMENTAL PROCEDURES |
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Materials-- Recombinant bIGFBP-2 was transiently expressed in the COS-1 (ATCC:CRL 1650) monkey kidney cell line and purified from medium conditioned by the transfected cells as described previously (33). Receptor grade IGF-I and IGF-II were the kind gifts of GroPep Pty. Ltd. (Adelaide, Australia). Radiolabeled 125I-IGF-I and 125I-IGF-II peptides were prepared to a specific activity of approximately 3 kBq/mol as described previously (34). Carrier-free Na125I was purchased from Amersham Pharmacia Biotech (Sydney, Australia). Reverse-phase HPLC columns were purchased from Brownlee Lab (Santa Clara, CA) and Amrad (Sydney, Australia). Pre-siliconized tubes (Sorenson BioScience, Inc., Salt Lake City, UT) were used for the collection of fractions during chromatography. All HPLC was carried out using Waters 510 solvent pumps, a Waters 490 4-channel absorbance detector (Millipore-Waters, Lane Cove, New South Wales), and a Perkin-Elmer LS4 fluorescence spectrometer (Scoresby, Victoria, Australia). The Waters Maxima software package was used to control solvent gradients and for data collection. HPLC-grade acetonitrile was purchased from Merck (Kilsyth, Victoria, Australia) and trifluoroacetic acid from Sigma-Aldrich (Castle Hill, New South Wales, Australia). All other reagents were analytical grade. Nitrocellulose filters were purchased from Schleicher & Schüll (Dassel, Germany). BIAcore reagents and supplies including CM5 sensor chips, HEPES-buffered saline (HBS), amine coupling reagents, N-ethyl-N'-[(dimethylamino)propyl]carbodiimide, N-hydroxysuccinimide, and ethanolamine were kindly provided by Pharmacia & Upjohn AB, Preclinical Research, Stockholm, Sweden, or were purchased from Amrad, Melbourne, Australia.
Mutagenesis--
A 344-base pair PstI-SmaI
fragment of the bIGFBP-2 pXMT2 based expression vector pGF8 (33) that
encompassed the Tyr-60 region was subcloned into the multiple cloning
site of the phagemid vector Bluescript pBKS() (Stratagene).
Mutagenesis was carried out by the Kunkel method (35) with the
MutaGeneTM Phagemid In Vitro Mutagenesis Version
2 kit (Bio-Rad, Regents Park, New South Wales, Australia). Six
mutagenic oligonucleotides were synthesized by Bresatec Ltd.,
Thebarton, South Australia, Australia. A summary of the mutagenic
oligonucleotide sequences and the resulting amino acid sequence changes
in bIGFBP-2 are shown in Fig. 2. For
screening purposes, the mismatch oligonucleotides also disrupted the
5'-GTAC-3' RsaI endonuclease site between the codons GTG and
TAC of Val-59 and Tyr-60. Therefore, positive clones were identified by
diagnostic RsaI digestion followed by polymerase chain
reaction DNA sequencing (36) using the sequencing primers 5'-GTTTTCCCAGTCACGAC-3' and 5'-CACACAGGAAACAGCTATGACCATG-3' which were
complementary to flanking sequences at the insertion site of pBKS(
).
Finally, pGF8 variants harboring the Tyr-60 region mutants were
regenerated by subcloning the mutagenized
PstI-SmaI fragments into the parent vector pGF8.
The expression vector integrity and correct base changes were confirmed
by RsaI restriction analysis and dye terminator DNA
sequencing (PRISM, Applied Biosystems, Victoria, Australia) using the
primer 5'-CTCGCCGTTGTCTGCAACCTGCTCCGGG-3'. bIGFBP-2 mutants were
expressed in COS-1 cells and purified as described previously (33).
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Analysis of the Mutant bIGFBP-2 Analogs-- Lyophilized samples of approximately 10 µg of each bIGFBP-2 mutant were submitted for electrospray mass spectrometry. The analysis was carried out on a Perkin-Elmer SCI-EX API-300 triple quadrupole mass spectrometer at the Australian Research Council electrospray mass spectrometry unit, Adelaide. Peptide concentrations were accurately quantified by both reverse-phase HPLC as described (32) and by amino acid analysis on an AminoQuant II/M High Sensitivity Instrument from Hewlett-Packard, Waldbronn, Germany (37), using the orthophthalaldehyde 9-fluorenylmethyl chloroformate two-stage derivatization procedure.
SDS-PAGE and Western Ligand Blot-- bIGFBP-2 samples (200 ng/lane) were electrophoresed on discontinuous 12.5% SDS-polyacrylamide gels under nonreducing conditions (38). The samples were either stained with silver (39) and quantified by densitometry (Molecular Dynamics) or transferred onto nitrocellulose filters for Western ligand blotting (40). The level of 125I-IGF-II binding to the bIGFBP samples on the filter was visualized and quantified on a PhosphorImager (Molecular Dynamics).
Circular Dichroism-- CD spectra were recorded using a Jasco J720 spectropolarimeter equipped with a PTC-348W1 Peltier Type Temperature Controller set to 20 °C. bIGFBP-2 samples were adjusted to a final concentration of 0.25 mg/ml with 10 mM sodium phosphate, 60 mM NaCl, pH 7.4, and placed in a quartz cuvette with a path length of 1 mm. Spectra were recorded from 250 to 190 nm with a step resolution of 0.2 nm and a scanning speed of 20 nm/min. The response time was set to 1 s and the bandwidth was 0.5 nm. Each spectrum is the average of five accumulated scans.
Soluble IGFBP Assay-- The relative IGF binding affinities of the bIGFBP-2 mutants were determined at equilibrium by charcoal binding assay, essentially as described previously (41) with the following modifications. The assay buffer pH was 7.4 and the IGFBP/IGF-tracer incubation period was 24 h at room temperature. Each bIGFBP-2 concentration was assayed in triplicate, and the total analysis was performed twice. In assays containing 125I-IGF-I, approximately 7,000 cpm/tube were used, and the nonspecific binding was 6% of the total radioactivity added. Similarly, 10,000 cpm/tube of 125I-IGF-II were used with a nonspecific binding of 8% of the total radioactivity added. The experimental data were fitted to a sigmoidal dose-response model with variable slope using GraphPad Prism (GraphPad Inc., San Diego).
BIAcore-- All BIAcore analyses were carried out with IGF as the immobilized ligand. Covalent attachment of either IGF-I or IGF-II to the CM5 biosensor chip was achieved by the amine coupling method (42). Briefly, IGF (12.5 µg/ml in 50 mM sodium acetate, pH 4.7) was injected onto the activated CM5 surface at 5 µl/min with HBS (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, pH 7.4) as the running buffer. Residual binding sites were quenched with ethanolamine. The multichannel capability of the BIAcore 2000 was used to generate channels with either IGF-I or IGF-II surfaces (prepared to final resonance values of approximately 60 to 120 RU above the resonance value of the activated but underivatized chip) as well as a reference surface to which no IGF was bound. In a kinetic study, bIGFBP-2 and bIGFBP-2 mutants (5-100 nM in HBS, n = 6 per bIGFBP-2 species) were injected for 5 min at a flow rate of 40 µl/min with HBS as the running buffer. The dissociation phase, initiated by switching from the stream of the bIGFBP-2 sample to HBS, was carried out over a period of 10 min. The IGF surfaces were regenerated by a 90-s injection of 0.1 M HCl. Due to the large number of bIGFBP-2 samples and the extended length of the experiment, samples were injected in random order, and the experiment was carried out twice on two different chips rather than with duplicate samples on a single chip. The apparent analyte association and dissociation rates were derived by fitting the experimental data to either a one-site (IGF-I) or two-site (IGF-II) association model and a two-site dissociation model with the BIAevaluation software (version 2.1) supplied with the instrument.
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RESULTS |
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Protein Characterization--
The Tyr-60 region bIGFBP-2 mutant
constructs were sequenced in the expression plasmid pGF8, and the
expected DNA sequences were obtained. Transient expression of bIGFBP-2
mutants in transfected COS-1 cells yielded approximately 400 µg of
each purified protein from 0.5 liters of conditioned medium. All of the
bIGFBP-2 mutants migrated as single bands of the same size as wild-type
bIGFBP-2 when run on nonreducing SDS-PAGE (Fig.
3a). A minor band
corresponding to bIGFBP-2 dimer was also evident in the SDS-PAGE
analysis (Fig. 3, a and b). When analyzed by
electrospray mass spectrometry, the observed mass of each mutant with
the exception of Tyr-60 Ala corresponded with the
sequence-predicted mass to within 3 mass units. Tyr-60
Ala bIGFBP-2
possessed a mass that was 18 mass units greater than expected, possibly
due to methionine oxidation. Electrospray mass spectrometry also
indicated that in each of the bIGFBP-2 and bIGFBP-2 mutant samples
there was a small but consistent contamination (approximately 5%) with
a species that was 355 mass units greater than the predicted mass. When
the N-terminal sequence of the minor contaminant of wild-type bIGFBP-2
was determined, the larger species was identified as a mis-processed
form of bIGFBP-2. The N terminus of the contaminant was
Gly-Ala-Arg-Ala, corresponding to the last four residues of the leader
peptide prior to the normal cleavage site (43) of mature bIGFBP-2.
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Circular Dichroism--
The secondary structure composition of the
bIGFBP-2 mutants were compared with wild-type bIGFBP-2 using CD
spectroscopy. An overlay of the far UV spectra of Val-59 Ala,
Tyr-60
Ala, Tyr-60
Phe bIGFBP-2, and wild-type bIGFBP-2 (Fig.
4) shows that the spectra of these
engineered bIGFBP-2 analogs were essentially the same as wild-type
bIGFBP-2. Any slight deviation from the CD spectra of bIGFBP-2 could be
explained by minor differences in protein concentration. The CD spectra
of Thr-61
Ala, Pro-62
Ala, and Arg-63
Ala bIGFBP-2 were
also very similar to wild-type bIGFBP-2 (not shown; these spectra were
omitted for clarity).
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Western Ligand Blots--
The 125I-IGF-II binding by
the bIGFBP-2 mutants was analyzed by Western ligand blot (Fig.
3b). Val-59 Ala, Tyr-60
Ala, and Tyr-60
Phe
bIGFBP-2 (Fig. 3b, lanes 2, 3 and 4) showed
reduced 125I-IGF-II binding compared with wild-type
bIGFBP-2 (Fig. 3b, lane 1). An estimate of the relative
binding of each bIGFBP-2 mutant was provided by direct comparison of
the band intensities from the ligand blot and the silver-stained gel.
After the bound 125I-IGF-II radioactivity corresponding to
each bIGFBP-2 species was quantified by PhosphorImager analysis (Fig.
3b) and corrected for the amount of peptide present on the
silver-stained gel (Fig. 3a), it was estimated that Val-59
Ala, Tyr-60
Ala, and Tyr-60
Phe bIGFBP-2 retained
approximately 40, 9, and 15% of the 125I-IGF-II bound by
bIGFBP-2 respectively. In contrast, Thr-61
Ala, Pro-62
Ala, and
Arg-63
Ala bIGFBP-2 (Fig. 3b, lanes 5-7) all bound
125I-IGF-II to a similar extent to bIGFBP-2 (Fig. 3b,
lane 1).
Solution IGF Binding Assay--
The IGF binding abilities of the
bIGFBP-2 mutants were characterized in solution binding assays (Fig.
5, Table
I). Alanine substitution for Val-59,
Thr-61, Pro-62, and Arg-63 of bIGFBP-2 did not significantly alter the
half-maximal binding concentration (EC50) of bIGFBP-2 for
either 125I-IGF-I (Fig. 5a) or
125I-IGF-II (Fig. 5b). In contrast both Ala and
Phe substitution for Tyr-60 of bIGFBP-2 resulted in a significant
increase in the EC50 for both 125I-IGF-I
(Tyr-60 Ala = 3.4-fold and Tyr-60
Phe = 4.7-fold) and
125I-IGF-II (Tyr-60
Ala = 2.2-fold and Tyr-60
Phe = 3.3-fold). The maximal 125I-IGF binding of the
bIGFBP-2 mutants was similar to wild-type bIGFBP-2 with 60% of added
IGF tracer for 125I-IGF-I and 47% of added IGF tracer for
125I-IGF-II. An exception was 125I-IGF-II
tracer binding to Tyr-60
Phe bIGFBP-2, where the maximal binding
was 37%, a reduction by
with respect to wild-type bIGFBP-2
(Table I).
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BIAcore Analysis-- Kinetic analyses of the association and dissociation of bIGFBP-2 and the bIGFBP-2 mutants with immobilized IGF-I and IGF-II were carried out in the BIAcore. Fig. 6, a and b, shows a representative subset of the sensorgram data (for qualitative comparison) that was used to generate the kinetic constants summarized in Table II. The interactions between all of the bIGFBP-2 peptides and both IGF-I and IGF-II were difficult to resolve to a single binding site model. In the case of IGF-I interactions, a single apparent association constant and two apparent dissociation constants produced the best fit of the sensorgram data. In contrast, two apparent association and two apparent dissociation constants were necessary for modeling bIGFBP-2 interactions on the IGF-II biosensor surface. The absolute values of the apparent kinetic constants for the IGFBP/IGF interactions (kon, koff, and KD, Table II) varied by up to 25% of the mean value between the two biosensor chips used in this study. However, the ranking of the bIGFBP-2 mutants relative to bIGFBP-2 (i.e. the fold differences in kon, koff, and KD, Table II) were the same on both biosensor chips used in this study.
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DISCUSSION |
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In this study, the role of the Tyr-60 region of bIGFBP-2 in IGF
binding has been investigated by alanine screening mutagenesis. In our
chemical modification study (32), Tyr-60, which lies in the N-terminal
cysteine-rich domain of bIGFBP-2, was shown to be protected from
iodination when either IGF-I or IGF-II was bound. The high degree of
sequence homology in the Tyr-60 region of the whole IGFBP family (Fig.
1) suggested that other residues in this vicinity might also play a
role in IGF binding. To investigate this possibility, Val-59 Ala,
Tyr-60
Ala, Tyr-60
Phe, Thr-61
Ala, Pro-62
Ala and
Arg-63
Ala bIGFBP-2 were recombinantly expressed, purified, and
characterized for changes in IGF affinity. The only bIGFBP-2 mutants
that were observed to exhibit significantly reduced affinity for IGF-I
and IGF-II using a variety of complementary analyses were Tyr-60
Ala and Tyr-60
Phe bIGFBP-2. All of the Tyr-60 region bIGFBP-2
mutants produced essentially the same CD spectra (Fig. 4) and all ran
as single bands at the same molecular weight as bIGFBP-2 in SDS-PAGE
analysis (Fig. 3). Therefore, any changes in the IGF binding
characteristics of the bIGFBP-2 mutants were considered to be due to
the loss of side chain interactions at the mutagenic site in question
and not to gross changes in protein structure.
There was good agreement between the three functional analyses that
were used to investigate the IGF binding abilities of the bIGFBP-2
mutants. Western ligand blot (Fig. 3b), solution binding
assays (Fig. 5, Table I), and the BIAcore analyses (Fig. 6, Table II)
all indicated that both Ala and Phe substitution for Tyr-60 resulted in
a bIGFBP-2 molecule with reduced affinity for IGFs. However, some
differences were noted. For example, Val-59 Ala bIGFBP-2 bound
125I-IGF-II at a visibly reduced level compared with
bIGFBP-2 in the Western ligand blot (Fig. 2), yet bound
125I-IGFs as well as bIGFBP-2 in the solution binding assay
(Fig. 4, Table I). Similarly, the decrease in 125I-IGF-II
binding of Tyr-60
Ala bIGFBP-2 was far more apparent in the Western
ligand blot (Fig. 3b) than in the solution binding assay
(Fig. 4, Table I).
Direct measurement of the association and dissociation kinetics of the
IGFBP-2/IGF interaction in the BIAcore experiments could explain the
observed differences between the Western ligand blot and the solution
binding assay results. Whereas Val-59 Ala and Tyr-60
Ala
bIGFBP-2 dissociated 2.3- and 6.2-fold more rapidly from the IGF-II
biosensor surface than bIGFBP-2, respectively, both also associated
with this surface approximately 2-fold more rapidly than bIGFBP-2.
Under the equilibrium conditions of the solution binding assay, the
increased dissociation rates of Val-59
Ala and Tyr-60
Ala
bIGFBP-2 were offset by the increases in the association rate. However,
in the Western ligand blot, nonspecifically bound
125I-IGF-II was washed from the filter by buffer
replacement, and so the possibility of IGF and IGFBP-2 reaching a
binding equilibrium was prevented. Therefore, we propose that the
Western ligand blot ranked the bIGFBP-2 mutants with respect to their
relative dissociation rates, whereas the solution binding assay ranked
the bIGFBP-2 mutants according to their overall affinities.
Insight into the reduced IGF binding affinity of Tyr-60 Ala and
Tyr-60
Phe bIGFBP-2 was provided by BIAcore analysis. Substitution
of Tyr-60 of bIGFBP-2 with Ala and Phe produced very different changes
in the kinetics of IGF interactions. Tyr-60
Ala bIGFBP-2 associated
with and dissociated from IGF biosensor surfaces more rapidly than
bIGFBP-2. In contrast, Tyr-60
Phe bIGFBP-2 exhibited a reduced rate
of association with, and an increased rate of dissociation from, IGF
biosensor surfaces compared with bIGFBP-2. Interestingly, Tyr-60
Ala and Val-59
Ala bIGFBP-2 exhibited very similar kinetics on the
IGF-I and IGF-II biosensor surfaces. The enhanced association and
dissociation kinetics of Val-59
Ala bIGFBP-2 with immobilized IGF
suggests that this mutation produced subtle changes to the structure of
the IGF-binding site of bIGFBP-2, without detriment to the net energy
of IGF binding. In contrast, Tyr-60
Ala bIGFBP-2 exhibited a net
reduction in the energy of IGF binding due to large increases in the
dissociation rate from IGF surfaces. Therefore, the shape and volume of
the side chains of Val-59 and Tyr-60 may help to define the IGF-binding site of bIGFBP-2. However, the aromatic function and the hydrogen bonding potential of Tyr-60 are clearly the most significant
contributors to the stability of the bIGFBP-2·IGF complex. The
replacement of Tyr-60 with Phe was anticipated to be the most subtle
mutation, with the aromatic side chain packing maintained and the loss
of the tyrosyl hydroxyl group the only change. Surprisingly, Tyr-60
Phe bIGFBP-2 exhibited the lowest affinities for IGFs, thus providing
further evidence to suggest that Tyr-60 participates in hydrogen
bond(s) that stabilize IGF interactions.
In circulation, IGFBPs bind IGFs with a 1:1 stoichiometry (reviewed in Ref. 7), and therefore a single site kinetic model should provide a valid approximation of the IGFBP/IGF interaction. Indeed, the apparent affinity constants that were calculated with the derived association and dissociation constants of bIGFBP-2 and the IGF-I and IGF-II surfaces (0.5 nM for IGF-I and 0.2 nM for IGF-II) correspond very well with published constants generated by competition solution binding assays (44, 45). Yet, the interactions of bIGFBP-2 and the bIGFBP-2 mutants with immobilized IGFs deviated from pseudo-first order kinetics in the BIAcore (Fig. 6). Multiple phase kinetics for single site interactions can be due to artifacts of the BIAcore assay conditions (46, 47). In this study, steps have been taken to address some of the common artifactual causes for multiple phase kinetics. Thus, very low density IGF biosensor surfaces (60-120 RU) and moderate flow rates (40 µl/min) have been deliberately used to minimize mass transfer limitations and the steric masking of binding sites. We have used amine coupling to immobilize IGFs in this study, and this strategy can lead to a mixed population of ligand on the biosensor surface with a range of different affinities for the analyte (47). However, the same non-pseudo-first order kinetic behavior was observed when immobilized hIGFBP-3 was analyzed with free IGF (48). It is therefore possible that the multiple association and dissociation phases present in IGF/IGFBP sensorgrams reflect a physical characteristic of the protein interaction such as multiple step binding. It is interesting to note that Scatchard analysis of competitive solution binding assays have also shown that IGFBPs may exhibit both low and high affinity binding sites for IGFs under some conditions (49, 50).
Overall, the effects of mutagenesis in the Tyr-60 region of bIGFBP-2
were more severe for interactions with IGF-I than IGF-II. For example,
Tyr-60 Phe bIGFBP-2 bound to the IGF-I biosensor surface with a
8.4-fold lower affinity than bIGFBP-2, whereas it bound to the IGF-II
biosensor surface with a 4.0-fold lower affinity than bIGFBP-2. This
corresponds well with our earlier findings that iodo-bIGFBP-2 exhibited
an 8-fold reduction in apparent affinity for IGF-I compared with a
4-fold reduction in apparent affinity to IGF-II (32). The high degree
of structural similarity between IGF-I and IGF-II (9) suggests that
both IGF molecules interact with IGFBPs through similar side chain
contacts. Mutagenic support for this idea can be seen in the similar
reduction in IGFBP affinity of [Arg3]IGF-I and its
structural homolog [Arg6]IGF-II (16). The
disproportionate sensitivity of the Tyr-60 bIGFBP-2 mutants toward
IGF-I binding, in addition to the natural preference of bIGFBP-2 for
IGF-II (4), suggests that the bIGFBP-2·IGF-II complex contains
additional points of molecular interaction that are absent in the
bIGFBP-2·IGF-I complex. Indeed, in a recent truncation study (33),
mutagenic removal of 62 amino acid residues from the C terminus of
bIGFBP-2 resulted in a dramatic loss of IGF binding. More importantly,
this bIGFBP-2 mutant had lost all binding preference for IGF-II over
IGF-I. It was thus concluded that the C-terminal cysteine rich domain
of bIGFBP-2 contained determinants of IGF-II binding
specificity.
The ability of Tyr-60 to form a hydrogen bond that is important for IGF
binding is evident by the reduced affinity of both Tyr-60 Ala and
Tyr-60
Phe bIGFBP-2 for IGF-I and IGF-II. Moreover, the presence of
an aromatic function at position 60 of bIGFBP-2 reduces the rate of
formation of bIGFBP-2·IGF complex but also enhances the stabilization
of the complex. This study raises the question as to whether the
hydrogen bond acceptor of Tyr-60 is within the bIGFBP-2 molecule or
within the IGF molecule. We propose that Tyr-60 is located in the IGF
binding interface of bIGFBP-2 and that modification or mutagenic
replacement of Tyr-60 directly disrupts contacts between IGF and
bIGFBP-2. However, the alternative possibility that Tyr-60 modification
and mutagenic replacement indirectly affects IGF binding for example,
by preventing a change in conformation that is necessary for high
affinity IGF binding, has not yet been eliminated. The simplest
interpretation of our earlier observation that iodination of Tyr-60 can
be blocked by the formation of an bIGFBP-2·IGF complex supports a
direct interaction between Tyr-60 of bIGFBP-2 and IGF. Clearly,
distinction between these two models must await biophysical
characterization of the IGFBP-2·IGF complex.
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ACKNOWLEDGEMENTS |
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The amino acid analyses were performed by Kristina Zachrisson, Pharmacia & Upjohn, Stockholm. G. D. H. would especially like to thank Professor Björn Nilsson, Dr. Göran Forsberg, Dr. Cornelia Oellig, Dr. Claes Andersson, and all of the members of the Biochemistry Section, Pharmacia & Upjohn AB for their help and enthusiasm.
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FOOTNOTES |
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* The CD and BIAcore work was supported by an Australian Society for Biochemistry and Molecular Biology Fellowship (to G. D. H.), a University of Adelaide Travel Award (to G. D. H.), and conducted in part at Pharmacia & Upjohn AB, Stockholm.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF074854.
¶ Recipient of a Ph.D. Scholarship from the Cooperative Research Center for Tissue Growth and Repair.
To whom correspondence should be addressed: Dept. of
Biochemistry, University of Adelaide, North Terrace, Adelaide, South Australia 5005, Australia. Tel.: 61-8-8303-5218; Fax: 61-8-8303-4348; E-mail: jwallace{at}biochem.adelaide.edu.au.
1 The abbreviations used are: IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; RU, resonance units; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
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