From the Department of Pharmacology, New York
University School of Medicine, New York, New York 10016, the
¶ Department of Biochemistry and Biophysics and the Johnson
Foundation, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104-6059, and the § Division of
Bioengineering and Environmental Health, Harvard-Massachusetts
Institute of Technology, Division of Health Sciences and Technology,
Cambridge, Massachusetts 02139
Received for publication, July 20, 2000, and in revised form, October 30, 2000
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ABSTRACT |
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Fibroblast growth factors (FGFs) constitute a large
family of heparin-binding growth factors with diverse biological
activities. FGF9 was originally described as glia-activating
factor and is expressed in the nervous system as a potent
mitogen for glia cells. Unlike most FGFs, FGF9 forms dimers in solution
with a Kd of 680 nM. To elucidate the
molecular mechanism of FGF9 dimerization, the crystal structure of FGF9
was determined at 2.2 Å resolution. FGF9 adopts a The mammalian fibroblast growth factor
(FGF)1 family contains at least
22 distinct polypeptides (FGF1-FGF22) that are expressed in a specific
spatial and temporal pattern (1-5). FGFs play important roles in
numerous physiological and pathological processes (1-5). Members of
the FGF family share between 10 and 55% sequence identity (6). Crystal
structures of FGF1 (7), FGF2 (7-9), and FGF7 (10), have revealed a
common core FGF structure consisting of three copies of a four-stranded
FGF9 (glia-activating factor) was purified as a heparin-binding,
secreted glycoprotein from cultured human glioma cell line NMC-G1 (11).
FGF9 shows 30% sequence identity with the prototypical FGF family
members, FGF1 and FGF2 (12). Purified FGF9 is mitogenic for many types
of cultured cells including glia cells, oligodendrocyte type 2 astrocyte progenitor cells, smooth muscle cells, pheochromocytoma PC12
cells, and BALB/3T3 fibroblasts (11). Because FGF9, unlike FGF1 and
FGF2, has no effect on human umbilical vein endothelial cells, it is
suggested that FGF9 may have a unique receptor specificity (11). In
fact, biochemical studies performed utilizing various soluble
FGFR-alkaline phosphatase fusion proteins and genetically engineered
cells expressing different full-length FGFRs have demonstrated that
FGF9 binds preferentially to the IIIc form of FGFR3 (13-15).
FGF9, like prototypical FGFs, does not have a typical secretory signal
peptide. Yet, it is still glycosylated and efficiently secreted from
transfected mammalian cells (12). N-terminal sequencing of secreted
FGF9 shows that it is missing only the initiation methionine (12, 16).
It has been suggested that FGF9 secretion is mediated via a noncleaved
signal sequence consisting of the first 33 residues (17, 18). We have
expressed and purified truncated FGF9 (residues 35-208) in
Escherichia coli and observed that it dimerizes in solution.
To elucidate the molecular mechanism of FGF9 dimerization, the crystal
structure of FGF9 was determined. In the crystal, FGF9 forms a 2-fold
dimer in which a large surface area is buried. This suggests that the
dimer observed in the crystal structure also represents the dimeric
FGF9 species detected in solution. Analysis of the dimer interface
reveals interactions between the N- and C-terminal segments outside of
the Protein Expression and Purification--
DNA fragments generated
by polymerase chain reaction of human FGF9 cDNA (encoding for
residues 35-208) were subcloned into the pET-28a bacterial expression
vector using NcoI and HindIII cloning sites.
Following transformation of the BL21 (DE3) bacterial strain, cells
containing the FGF9-expression plasmid were induced with 1 mM isopropyl-1-thio- Crystallization and Data Collection--
Crystals of FGF9 were
grown by vapor diffusion at 20 °C using the hanging drop method. 2 µl of protein solution (1.4 mg/ml in 25 mM HEPES-NaOH, pH
7.5, and 150 mM NaCl) were mixed with 8 µl of various
crystallization buffer conditions of Crystal Screen kits (Hampton
Research). Condition 48 (kit I) (2 M ammonium phosphate, 0.1 M Tris-HCl, pH 8.5) produced diffraction quality
crystals. FGF9 crystals belong to the tetragonal space group
I4122 with unit cell dimensions a = b = 87.82 Å, c = 115.64 Å. There is
one molecule of FGF9 in the asymmetric unit with a solvent content of
~56%. Diffraction data were collected from a flash frozen (in a dry
nitrogen stream using mother liquor containing 20% glycerol as
cryo-protectant) crystal on a CCD detector at beamline X4A at the
National Synchrotron Light Source, Brookhaven National Laboratory. Data
were processed using DENZO and SCALEPACK (19).
Structure Determination and Refinement--
A molecular
replacement solution was found for one copy of FGF9 in the asymmetric
unit using the program AmoRe (20) and the structure of
FGF42 as the search model.
Simulated annealing and positional/B factor refinement were performed
using CNS (21). Bulk solvent and anisotropic B factor corrections were
applied. Model building into 2Fo Matrix-assisted Laser Desorption Ionization Mass Spectrometry
Analysis of FGF9--
Mass spectral analysis of FGF9 was completed by
diluting 1:10 of an aqueous 10 µM stock solution of FGF9
with a saturated solution of sinapinic acid in 30% acetonitrile. The
sample was spotted on a seeded surface and dried under a stream of
nitrogen (23). MALDI-MS were acquired in the linear mode using delayed extraction with a Voyager Elite reflectron time-of-flight instrument (PerSeptive Biosystems, Framingham, MA) with a 337-nm laser. Spectra were acquired using instrument settings described previously (24). Mass
spectra were calibrated externally with myoglobin and bovine serum albumin.
Sedimentation Equilibrium Ultracentrifugation
Analysis--
Sedimentation equilibrium experiments employed an XL-A
analytical ultracentrifuge (Beckman). Samples (in 25 mM
HEPES, pH 7.5, containing 1.5 M NaCl) were loaded into
six-channel epon charcoal-filled centerpieces, using quartz windows.
Experiments were performed at 20 °C, detecting at 235 and 278 nm,
using four different speeds (12,000, 18,000, 24,000, and 30,000 rpm.).
The program SEDNTERP was used to estimate the solvent density (1.062 g/ml) from its components and the partial specific volume of FGF9
(0.732 ml/g) from its amino acid composition (25). Experiments were
performed with a range of FGF9 concentrations ranging from 1.6 to 26 µM at the speeds mentioned above.
The Optima XL-A Data Analysis Software package (Beckman/MicroCal) was
used for global fits of the data to self-association models using nine
data sets with 278 nm detection and (independently) nine data sets with
235 nm detection. Identical results were obtained for each group of
nine data sets. Reasonable global fits to the data could be obtained
only with a simple dimerization model, as discussed under "Results
and Discussion." Goodness of fit was judged by the occurrence of
small, randomly distributed, residuals for the fits, as shown in Fig.
2B.
Structure Determination--
The first 33 N-terminal residues of
FGF9 were deleted because they are implicated only in secretion and not
FGFR binding (17, 26). Truncated FGF9 (residues 35-208) was expressed
in E. coli and purified to homogeneity (see "Experimental
Procedures"). Crystallization trials with FGF9 produced tetragonal
crystals with one molecule per asymmetric unit. The crystal structure
of FGF9 was solved by molecular replacement (see "Experimental
Procedures") and refined at 2.2 Å resolution with an R
value of 20.7% (free R value of 23.1%). The atomic model
for FGF9 consists of one FGF9 molecule (residues 52-208), two
phosphate ions, and 68 water molecules. Data collection and refinement
statistics are given in Table I.
Description of the Structure--
As anticipated on the basis of
sequence alignment, FGF9 adopts a
The most striking structural differences between FGF9 and the
prototypical FGFs are the conformations of the N- and C-terminal regions outside of the FGF9 Dimers and Autoinhibition--
As shown in Fig.
2A, the elution position of FGF9
on the size exclusion chromatography column is dependent on the
concentration at which it is loaded. When loaded at 2 mg/ml (100 µM), FGF9 elutes at a position corresponding to a
molecular mass of ~40 kDa, indicating that FGF9 forms dimers. By
contrast, when loaded at a concentration of 8 µg/ml (0.4 µM), FGF9 elutes at 32 ml, consistent with its monomeric
molecular mass (20 kDa). When concentrations between 8 µg/ml and 2 mg/ml are loaded, the elution position of FGF9 is intermediate,
suggesting that FGF9 reversibly dimerizes with a Kd
in the micromolar range. This was confirmed quantitatively by
sedimentation equilibrium analytical ultracentrifugation studies (Fig.
2B), which showed that FGF9 dimerizes with a
Kd of 680 nM. Sedimentation of FGF9 was
analyzed for a series of solutions at concentrations from 1.6 to 26 µM, as described under "Experimental Procedures."
Attempts to fit the data to a single species suggested that FGF9
self-associates, and only a simple dimerization model could be fitted
globally to multiple data sets collected at different concentrations
and speeds. The global fit to the data shown in Fig. 2B
represents a model in which FGF9 dimerizes with a Kd
of 680 nM. As seen above each graph, the residuals for this
fit are small and random for each individual data set.
Additional verification of the ability of FGF9 to dimerize was achieved
using MALDI-MS under conditions that do not interfere with
protein-protein interactions (30, 31). Using sinapinic acid as the
matrix, two FGF9 species are detected, with molecular masses of 20 and
40 kDa (Fig. 2C). Use of other matrix conditions, including
ferulic acid in tetrahydrofuran and 6-aza-2-thiothymine in ammonium
acetate, confirms that the dimer observed in the mass spectrum is not
simply an artifact of MALDI-MS sample preparation (data not shown) and
provides further support for FGF9 dimerization in solution.
Information regarding possible structural modes of FGF9 dimerization
can be gained by close inspection of the FGF9 crystal structure.
Analysis of the symmetry mates in the crystal reveals a 2-fold
crystallographic dimer that is particularly intimate (Fig.
3). The large buried surface area (2260 Å2) in the interface between molecules in this dimer is
compatible with our measured dissociation constant of 680 nM, suggesting that this is the dimer that occurs in
solution.
The dimer interface can be analyzed as the sum of two interfaces: an
interface outside the
The portion of the dimer interface inside the Receptor Binding Site and Specificity--
Receptor binding
specificity is an essential regulatory element of FGF responses and is
achieved through both primary sequence variations and alternative
splicing. Comparison of three different FGF-FGFR structures defined a
general binding interface for FGF-FGFR complexes that involves contacts
made by FGF to D2 and to the linker between D2 and the Ig-like domain 3 (D3) (32). It was also shown that specificity is achieved through
interactions between the FGF N-terminal (immediately preceding the
To provide a molecular basis for the specificity of FGF9 toward
FGFR3(IIIc), we superimposed the FGF9 structure onto the FGF2 structure
complexed with the ligand binding portion of FGFR1 consisting of D2 and
D3 (Fig. 6A). The majority of the
interactions in the FGF-FGFR interface can easily be accommodated with
minor adjustments of side chain rotamers. However, analysis of the
FGF9-FGFR1 interface identified two critical regions, the
At the primarily hydrophobic FGF9-D2 interface, three critical FGF9
residues (Tyr-67, Tyr-145, and Leu-188) are highly conserved among
different members of the FGF family (Fig. 6B). In the
FGF9-linker interface, Asn-146, also highly conserved among FGFs, is
expected to form hydrogen bonds with an FGFR-invariant arginine
(Arg-250 in FGFR1) in the D2-D3 linker region. In contrast, FGF9
residues predicted to be in the FGF9-D3 interface show little
conservation among FGFs. Close inspection of the FGF9-D3 interface at
the Heparin Binding Site--
The requirement of heparin/heparan
sulfate proteoglycans for FGF-induced FGFR dimerization is well
documented (34). Both FGFs and FGFRs are known to bind heparin (35).
The recent crystal structure of a ternary FGF2-FGFR1-heparin complex
has provided a mechanistic view of the process by which heparin aids
FGFs to induce FGFR dimerization (33). According to the proposed
"two-end" model, heparin interacts via its nonreducing end with the
heparin binding sites of FGF and FGFR and promotes the formation of a ternary 1:1:1 FGF-FGFR-heparin complex. A second ternary 1:1:1 FGF-FGFR-heparin complex is then recruited to the first ternary complex
via interactions of FGFR, FGF, and heparin in one ternary complex with
the FGFR in the adjoining ternary complex.
Within each ternary 1:1:1 FGF-FGFR-heparin complex, heparin makes
numerous contacts with the heparin binding residues of FGF and FGFR,
thereby increasing the affinity of FGF toward FGFRs. This provides the
molecular basis for the well documented heparin-dependent 1:1 FGF-FGFR interaction. In addition heparin also interacts with the
heparin binding site of the adjoining FGFR, thereby augmenting the weak
interactions of FGF and FGFR in one ternary complex with the FGFR in
the adjoining ternary complex. In the absence of heparin, these
interactions are not sufficient for sustained dimerization to occur.
Based on the model, differences in the primary sequences of heparin
binding sites of FGFs and FGFRs will effect
heparin-dependent FGF-FGFR binding affinity. Hence, each
FGF may require different heparin motifs (sulfation pattern and/or
length) to exert their optimal biological activities.
To analyze potential heparin binding sites of FGF9, a dimeric model for
FGF9-FGFR1 was generated by superimposing two copies of the FGF9
structure onto the two copies of FGF2 in the ternary FGF2-FGFR1-heparin
structure. As in the FGF2-FGFR1 structure, calculation of the
electrostatic surface potential identifies a positively charged canyon.
Two phosphate ions (provided by the crystallization buffer) are bound
in the predicted high affinity heparin binding sites of FGF9 (Fig.
7A). Because heparin or heparan sulfate moieties of cell surface proteoglycans are certainly longer than heparin decasaccharides used in the FGF2-FGFR1-heparin crystals (33), it is reasonable to suggest that natural heparins may interact
with FGFs beyond the well characterized low and high affinity heparin
binding sites. Indeed, close inspection of surface residues at the
periphery of the conventional FGF high affinity site led us to consider
two unreported potential sites: one at the very distal end of the
canyon and another that is more central to high affinity site. The
distal site, composed of Thr-81, Lys-121, and Glu-123, does not
coordinate a phosphate ion in the FGF9 structure (Fig. 7B).
However, the crystal structure of FGF42 reveals a sulfate
ion bound at this site by side chains of the corresponding FGF4
residues (Arg-103, Lys-142, and Lys-144). Thompson et al.
(36), who mutated several surface residues of FGF2 to identify
potential heparin binding sites, have provided further evidence for the
existence of a distal site. Mutation of Arg-81 in FGF2, which
corresponds to Glu-123 in FGF9, was found to cause a 2-fold reduction
in heparin binding.
In contrast, the "central" site does coordinate a phosphate ion via
the side chains of Tyr-163 and Arg-180 (Fig. 7B). Several other FGF9 residues in the vicinity of this phosphate ion may also
interact with heparin (Fig. 7B). Interestingly, several of these residues, including Tyr-163, are located in the highly divergent
Given the importance of FGF signaling in numerous biological processes,
it is likely that multiple levels of regulation exist to modulate FGF
activity. Besides FGF9 and FGF16, several other uncharacterized members
of the FGF family such as FGF17, FGF18, and FGF19 contain large N- and
C-terminal regions outside the -trefoil fold
similar to other FGFs. However, unlike other FGFs, the N- and
C-terminal regions outside the
-trefoil core in FGF9 are ordered and
involved in the formation of a 2-fold crystallographic dimer. A
significant surface area (>2000 Å2) is buried in the
dimer interface that occludes a major receptor binding site of FGF9.
Thus, we propose an autoinhibitory mechanism for FGF9 that is dependent
on sequences outside of the
-trefoil core. Moreover, a model is
presented providing a molecular basis for the preferential affinity of
FGF9 toward FGFR3.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-sheet, known as a
-trefoil fold. All FGFs contain N- and
C-terminal segments outside the
-trefoil core. However, the length
of these segments, especially the C termini, varies greatly among
different FGFs. In FGF1, FGF2, FGF4, and FGF7, the end of polypeptide
chain virtually coincides with the end of the
-trefoil fold.
Consequently, these FGFs have extremely short C-terminal segments. In
contrast, other FGFs, such as FGF3, FGF5, and FGF8, have long
C-terminal extensions. The functional relevance of these N- and
C-terminal extensions remains uncharacterized.
-trefoil core of each FGF9 protomer that are the driving force
for dimer formation. Interestingly, a major FGFR binding site becomes
occluded upon dimer formation. Thus, we propose that FGF9 regions
outside the
-trefoil core have an autoinhibitory role in FGF9
function. Furthermore, our modeling studies afford a structural basis
for the preferential affinity of FGF9 toward FGFR3 and for its specific heparin requirement.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-galactopyranoside for
5 h. The bacteria were then centrifuged and subsequently lysed in
a 25 mM HEPES-NaOH buffer (pH 7.5) containing 150 mM NaCl, 10% glycerol, and 5 mM EDTA
using a French press. FGF9 was found mainly in the soluble fraction and
precipitated with saturated ammonium sulfate solution (final
concentration, 50%) overnight at 4 °C. Following centrifugation,
the pellet was dissolved in a HEPES-NaOH buffer (pH 7.5) containing 1 M ammonium sulfate, centrifuged and diluted 1:5 prior to
loading onto a Source S column (Amersham Pharmacia Biotech). Bound FGF9
was eluted by a linear gradient of NaCl to 1 M in a 25 mM HEPES-NaOH (pH 7.5) buffer. Subsequent purification of
FGF9 was achieved by size exclusion chromatography on a Superdex 75 column (Amersham Pharmacia Biotech) equilibrated with a 25 mM HEPES-NaOH buffer (pH 7.5) containing 1.5 M
NaCl. Mass spectrometry of purified FGF9 confirmed the predicted
molecular mass of 20,169 daltons for FGF9.
Fc and Fo
Fc electron density maps was performed with
program O (22). The atomic model contains residues 52-208 of FGF9, two phosphate ions, and 68 water molecules. The average B factor is 18.5 Å2 for FGF9 molecule, 56.0 Å2 for phosphate
ions, and 24.0 Å2 for water molecules.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Summary of crystallographic analysis
-trefoil fold (Fig.
1). Superimposition of the C
traces located within the
-trefoil core of FGF9 with those belonging to
FGF1 and FGF2 gives a root-mean-square deviation of only 1.0 and 0.73 Å, respectively. Within the
-trefoil core, the major differences
between the FGF9 structure and the structures of FGF1 and FGF2 are the
1-
2 and
9-
10 loop conformations. These loops vary both in
length and sequence among the various members of the FGF family. In
FGF9, the
1-
2 loop is one residue shorter than the corresponding
loops of FGF1 and FGF2. In contrast, the
9-
10 loop in FGF9 is
longer by 4 and 6 residues than the corresponding loop in FGF1 and
FGF2, respectively. This loop bulges out from the main
-trefoil body
of FGF9. Two ordered phosphate ions are coordinated in the FGF9
structure; one is bound to the high affinity heparin-binding site at a
position similar to where sulfate ions/groups bind ligand in the
structures of free or heparin-bound FGF1 (27, 28) and FGF2 (8, 9, 29).
The other phosphate ion is bound by FGF9 residues, whose homologues in
FGF1 and FGF2 have not been implicated in sulfate ion coordination.
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Fig. 1.
Ribbon diagram of FGF9. Secondary
structure assignments were obtained with the program PROCHECK (37). The
strands of FGF9 are labeled according to the conventional strand
nomenclature for FGF1 and FGF2 (38). NT and CT
denote the N and C termini. This figure was created using the
programs Molscript (39) and Raster3D (40).
-trefoil core. In the crystal structures of
free FGF1 and FGF2, the region preceding the
-trefoil core is
disordered. However, in the FGF9 structure, part of this region is
ordered and forms an
helix (
N) (Fig. 1). Additionally, the entire C-terminal tail following the
-trefoil core is also ordered and contains a short helix (
C) (Fig. 1). The C-terminal region is
considerably longer in FGF9 (17 residues) than the C-terminal regions
of FGF1 (4 residues), and FGF2 (3 residues). Several interhelical interactions between the
N and
C helices contribute to the
ordering of the N- and C-terminal segments in FGF9.
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Fig. 2.
FGF9 dimerizes in solution.
A, 1 ml of purified FGF9 solutions of decreasing
concentrations were analyzed by size exclusion chromatography.
Black, 2 mg/ml; blue, 0.5 mg/ml;
green, 40 µg/ml; brown, 20 µg/ml;
red, 8 µg/ml. Arrows indicate the position of
the size standards: 67 kDa, albumin; 43 kDa, ovalbumin; 25 kDa,
chymotrypsinogen; 13.7 kDa, ribonuclease A. B, sedimentation
equilibrium analysis of FGF9 was performed with sample concentrations
ranging from 1.6 to 26 µM. Distribution of FGF9 in the
cell was determined by absorbance at 278 nm (at 12,000, 18,000, and
24,000 rpm) or 235 nm (at 18,000, 24,000, and 30,000 rpm). A total of
18 different data sets could be fit globally only to a simple
dimerization model in which FGF9 dimerizes with a Kd
of 680 nM (see text). Data for four different
concentrations are shown (1.6, 3.3, 6.6, and 13.2 µM),
each at three different speeds. Symbols correspond to data points, and
the curve through the symbols corresponds to the global best
fit. Residuals for this fit are plotted in the upper panel
for each data set and are both small and randomly distributed. Residual
plots have been arbitrarily displaced along the y axis for
clarity. C, MALDI-MS analysis of 1 pmol of FGF9 using
sinapinic acid as the matrix in 30% acetonitrile. Detected in the mass
spectrum are clear signals for the monomeric FGF9 at
m/z 20,169 and dimeric FGF9 at
m/z 40,230. No higher order oligomers are
observed.
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Fig. 3.
The 2-fold crystallographic FGF9 dimer.
A, ribbon diagram of the FGF9 dimer. The two FGF9 protomers
are colored orange and red, respectively.
B, space-filling model of the FGF9 dimer (same orientation
as in A). Coloring of the protomers is the same as in
A. FGF9 residues that participate in dimer formation are
colored only in one of the protomers. FGF9 residues are considered to
be in the dimer interface only if one pair of atoms (side chain or main
chain) has an inter-atomic distance of less than or equal to 3.8 Å. In
addition, dimer interface residues that are predicted to be in the
receptor interface are colored with respect to the FGFR region with
which they would interact. FGF9 residues that are predicted to interact
with D2 are colored green, residues that are predicted to
interact with the linker region are colored gray, and
residues that are predicted to interact with D3 are colored
cyan. C, space-filling representation of the
dimer interface. The dimer, shown in B, was rotated 90°
about the vertical axis as indicated, and one of the protomers
(red) was removed to improve visualization of the dimer
interface. Interface residues that are predicted to participate in FGFR
binding are labeled. This figure was created using Molscript and
Raster3D.
-trefoil core (Fig.
4A) and an interface inside the
-trefoil core (Fig. 4B). The interface outside the
-trefoil involves the
N and
C helices as well as the
respective loops that connect them to the main
-trefoil body. At
this interface, the aliphatic side chains of Leu-54, Ile-60, Leu-61,
Leu-200, and Ile-204 from one FGF9 protomer are in hydrophobic contact with the corresponding residues of the other protomer (Fig.
4A). A number of hydrogen bonds further strengthen this
interface. Sequence alignment of FGFs shows that most of the residues
that participate in this interface are conserved in FGF16, suggesting that FGF16 may also dimerize in solution (Fig.
5). Analogous residues in the remaining FGFs
are divergent, implying that dimerization of these ligands by a similar
mechanism is not likely.
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Fig. 4.
Detailed interactions at the FGF9 dimer
interface. A, stereo view of the portion of the dimer
interface outside the -trefoil core. B, stereo view of
the portion of the dimer interface inside the
-trefoil core. The
coloring of the protomers is the same as in Fig. 3. Oxygen atoms are
colored red, nitrogen atoms are blue, and carbon
atoms have the same coloring as the protomers to which they belong.
Only side chains of interacting residues are shown. Dotted
lines represent hydrogen bonds. At the right side of
each stereo pair, a view of the whole structure in the exact
orientation as in stereo view is shown, and the region of interest is
highlighted. This figure was created using Molscript and
Raster3D.
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Fig. 5.
Structure-based sequence alignment of
FGFs. Sequence alignment was performed using the CLUSTALW (41).
All of the FGFs used in this alignment are human. The location and the
length of the strands and
helices are shown on the
top of the sequence alignment. A period indicates
sequence identity to FGF9. A dash represents a gap
introduced to optimize the alignment. FGF9 residues that participate in
dimerization are colored red. In blue are FGF9
residues that constitute the conventional low and high affinity heparin
binding sites. FGF9 residues that localize to the periphery of the high
affinity heparin-binding site and are predicted to form the distal and
central heparin binding sites are colored green and
yellow, respectively.
-trefoil core involves
the
1 and
12 strands as well as the
8-
9 loop (Fig. 4B). Several hydrogen bonds fortify hydrophobic contacts
that are made at this interface (Fig. 4B). Surprisingly,
several FGF9 residues that participate in this interface are also
predicted to interact with Ig-like domain 2 (D2) of FGFR in an
FGF9-FGFR complex. As expected, these residues are highly conserved
among different FGFs. Still, solution dimers for other FGFs have not been reported, insinuating that these interactions alone are not sufficient to drive FGF dimerization. Therefore, interactions between
FGF9 protomers at the dimer interface outside the
-trefoil core are
essential in promoting FGF9 dimerization. These interactions must
facilitate the formation of the dimer interface within the
-trefoil
core and cooperate with this second interface for dimerization to
occur. In the process, critical receptor binding sites appear to become
occluded upon dimerization, suggesting that dimerization may serve as a
biologically relevant autoinhibitory mechanism. We propose that
promotion of dimerization by the N- and C-terminal regions outside the
-trefoil core in FGF9 is responsible for driving occlusion of
receptor binding sites in the
-trefoil core. Based on the sequence
conservation between FGF9 and FGF16, we propose that a similar
autoinhibitory mechanism can be extended to FGF16. Receptor binding
studies using C- and N-terminal deletion mutants of FGF9 and FGF16
should further elucidate the importance of these regions.
-trefoil core) and central regions with two loop regions in FGFR D3
that are subject to alternative splicing (32).
8-
9
loop (within the
-trefoil core) and the
N helix (at the N
terminus outside the
-trefoil core), that are sterically clashing
with the receptor (colored red in Fig. 6A). These
two regions of FGF9 would require major changes in backbone
conformation to allow an engagement with FGFR1 to occur.
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Fig. 6.
Mapping of receptor binding sites in
FGF9. A, a model for the FGF9-FGFR1 structure was
generated by superimposition of the C traces within the
-trefoil
of the FGF9 structure onto the corresponding C
traces of FGF2 in the
FGF2-FGFR1 structure. Orange, FGF9; green, D2;
cyan, D3; gray, linker region; red,
FGF9 regions that are in major clashes with FGFR1. B, stereo
view of the receptor binding sites on FGF9. FGF9 residues are colored
with respect to the FGFR regions with which they interact. FGF9
residues that interact with D2 are colored green, residues
that interact with the linker region are colored gray, and
residues that interact with D3 are colored cyan. FGF9
residues that interact with the
C'-
E loop in D3 of FGFR are
colored purple. Color coding for atoms is the same as Fig.
4. This figure was created using Molscript and Raster3D.
C'-
E loop region in D3 affords a potential explanation for
why FGF9 binds preferentially to FGFR3(IIIc) over FGFR1(IIIc). The
C'-
E loops of the two FGFRs differ at two positions.
Significantly, the residue corresponding to Val-316 in FGFR1 is an
alanine (Ala-313) in FGFR3. In the FGF2-FGFR1 structure, Val-316 makes
hydrophobic contacts with Tyr-73, Val-88, and Phe-93 in FGF2. Although
Tyr-73 is conserved in all FGFs including FGF9, Leu-130 and Val-135 in FGF9 replace Val-88 and Phe-93 in FGF2 (Fig. 6B). The larger
hydrophobic side chain of Leu-130 in FGF9 clashes sterically with
Val-316 in FGFR1, thus reducing the affinity of FGF9 toward
FGFR1(IIIc). Conversely, the bulkier Leu-130 side chain would better
engage the smaller side chain of Ala-313 in FGFR3(IIIc). Interestingly, two other FGF9 residues (Ile-98 and Ile-100) also fall in the vicinity
of Ala-313 in the FGF9-FGFR3 model. The aliphatic side chains of these
two residues may provide additional hydrophobic contacts with Ala-313
in FGFR3(IIIc) (Fig. 6B). Ile-98 and Ile-100 lie in strand
4, whose counterpart in FGF2 was shown to play a critical role in
determining ligand-receptor specificity in both FGF2-FGFR1 and
FGF2-FGFR2 complexes (32, 33).
View larger version (64K):
[in a new window]
Fig. 7.
Heparin binding sites in FGF9.
A, the dimeric model for a FGF9-FGFR1 structure was created
by superimposition of the C traces of two FGF9 structures onto the
C
traces of the two FGF2 molecules in the FGF2-FGFR1-heparin
structure. The surface charge distribution of this dimer at the
putative heparin binding canyon is shown at the right side
of A. With the exact orientation the molecular surface of
the dimer is shown at the left side of A. The
phosphate ions in the high affinity heparin-binding sites of FGF9
molecules are rendered in ball-and-stick. This figure was generated
with GRASP (42). B, residues that localize to the heparin
binding surface of FGF9 in the context of FGF9-FGFR1 dimer are mapped
onto the ribbon diagram of FGF9. FGF9 residues that localize to the
peripheries of the high affinity heparin binding site and are predicted
to form the distal and central heparin binding sites are underlined. A
phosphate ion is bound in the conventional high affinity heparin
binding sites. The other phosphate ion is bound in the central site.
Dotted lines represent hydrogen bonds. This figure was
created using Molscript and Raster3D.
9-
10 loop. Because bound sulfate ions at the high affinity
heparin binding site of FGFs often mimic the position of the sulfate
groups of heparin in heparin-bound FGFs, it is reasonable to suggest that this central site may indeed constitute a genuine heparin binding
site. Confirmation of the aforementioned hypothesis requires structural
determination of a ternary FGF9-FGFR3-heparin complex.
-trefoil main body. It is possible
that these regions may also possess some type of autoregulatory
activity. Structural delineation of such regions in FGFs may be
particularly significant for the design of FGF mimetics. FGF agonists
may be used therapeutically to accelerate wound healing or to induce
angiogenesis in pathological conditions such as angina and stroke.
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ACKNOWLEDGEMENTS |
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We thank C. Ogata for synchrotron beamline assistance and B. K. Yeh for critically reading the manuscript. Beamline X4A at the National Synchrotron Light Source, a Department of Energy facility, is supported by the Howard Hughes Medical Institute.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants DE13686 (to M. M.) and CA79992 (to M. A. L.).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.
To whom correspondence should be addressed. Tel.:
212-263-2907; Fax: 212-263-7133.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M006502200
2 M. Mohammadi, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: FGF, fibroblast growth factor; FGFR, FGF receptor; D2, immunoglobulin-like domain 2; D3, Immunoglobulin-like domain 3; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry.
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