(Received for publication, May 25, 1995; and in revised form, July 5, 1995)
From the
The importance of basic fibroblast growth factor (bFGF) in several pathophysiological processes has stimulated interest in the design of receptor antagonists to mitigate such effects. Of key importance in this connection is the characterization of the functional binding epitopes of the growth factor for its receptor. Based on peptide mapping and molecular dynamics calculations of the three-dimensional structure of basic fibroblast growth factor, we employed site-directed mutagenesis to investigate the effect of altering residues at positions 107, 109-114, and 96 on bFGF on receptor binding affinity. All muteins were cloned and expressed in Escherichia coli, purified to homogeneity employing heparin-Sepharose columns, and evaluated for receptor binding affinity. We found that replacement of residues at positions 107 and 109-114 by alanine or phenylalanine had little effect on receptor binding affinities compared with wild type bFGF, in agreement with previous evidence that bFGF residues 109-114 comprise a low affinity binding site. By contrast, substitution of Glu-96 with alanine yielded a molecule having about 0.1% of the affinity of the wild type bFGF. The affinity of the corresponding lysine and glutamine muteins was 0.3 and 10%, respectively, emphasizing the importance of a negative charge at this position. Our findings are consistent with the view that residues 106-115 on bFGF represent a low affinity binding site on bFGF. In addition, we identify Glu-96 as a crucial residue for binding to fibroblast growth factor receptor-1.
Basic fibroblast growth factor (bFGF) ()is a potent
mitogen for a wide variety of cell types of mesodermal and
neuroectodermal origin (Folkman and Klagsbrun, 1987; Gospodarowicz,
1974; Gospodarowicz et al., 1987; Burgess and Maciag, 1989).
It has been suggested that bFGF and other members of this family may
play a critical role in several pathophysiological processes, including
the growth of tumors (Folkman, 1985), wound healing response (Folkman
and Klagsbrun, 1987), and diabetic retinopathy (Sivalingam et
al., 1990). The discovery of the high affinity binding of bFGF to
heparin has accelerated its purification, characterization, and cloning
(Shing et al., 1984; Abraham et al., 1986). So far,
nine members of the FGF family have been identified, including acidic
FGF (aFGF; Jaye et al., 1986), bFGF (Abraham et al.,
1986), int-2 gene product (int-2; Dickson and Peters, 1987), hst/kFGF
(Delli-Bovi et al., 1987; Yoshida et al., 1987),
FGF-5 (Zhan et al., 1988), FGF-6 (Marics et al.,
1989), keratinocyte growth factor (KGF; Finch et al., 1989),
and the actin-binding protein hisactophilin (Habazettl et al.,
1992). DNA sequencing of members of this gene family has revealed that
they possess 30-55% identity at the primary amino acid sequence
level (Dionne et al., 1990).
Several three-dimensional structures of acidic and basic FGFs are available. These include two acidic forms (Zhu et al., 1991, 1993) and three basic forms (Zhu et al., 1991; Zhang et al., 1991; Eriksson et al., 1991). Baird et al.(1988) identified two important regions on bFGF for binding to FGFR1, comprising residues 24-68 and residues 106-115. These workers showed that the sequence 24-68 has high affinity for heparin. Hence, the region 106-115 (Fig. 2) was considered the putative receptor binding site on bFGF.
Figure 2:
A ribbon rendering of the energy refined
(see ``Experimental Procedures'') three-dimensional structure
of basic fibroblast growth factor. Amino acid side chains that mutated
in these studies are labeled and the rest are shown in the orange
ribbon diagram of the C backbone of bFGF. The N terminus of
the protein is shown in blue and the C terminus in red.
Recently, the binding site for heparin/heparin sulfate on bFGF was more explicitly defined (Thompson et al., 1994; Li et al., 1994). Through identification of key amino acids for heparin binding on bFGF based on its three-dimensional structure and site-directed mutagenesis, these studies indicate that pure electrostatic interactions contribute only 30% of the binding free energy to the ligand receptor interaction and that hydrophobic effects, as well as other noncovalent forces such as hydrogen bonding and van der Waals packing, contribute the bulk of the free energy for this binding reaction. Heparin, although it is not absolutely required for bFGF to bind to its receptor (Shi et al., 1993; Roghani et al., 1994; Pantoliano et al., 1994), increases the receptor affinity of bFGF (Roghani et al., 1994), protects bFGF from inactivation (Gospodarowicz and Cheng, 1986) and proteolytic degradation (Saksela et al., 1988; Sommer and Rifkin, 1989), and is essential for the mitogenic activity of bFGF stimulated cells (Yayon et al., 1991).
In addition, Springer et al.(1994) identified high and low affinity receptor binding surfaces on bFGF on the basis of site-directed mutagenesis and molecular modeling. Based on their data, Tyr-103, Leu-140, and Tyr-24 on bFGF contribute significantly to the primary, higher affinity binding interaction. They found that the FGFR binding site composed of amino acids Lys-110, Tyr-111, and Trp-114 (Baird et al., 1988), referred to as the putative receptor binding region, is a secondary, lower affinity.
Here we describe site-directed
mutagenesis studies on the putative receptor binding region and its
neighbors based on the three-dimensional structure of basic fibroblast
growth factor refined by molecular dynamics. This is in contrast to the
work of Thompson et al.(1994), Li et al.(1994), and
Springer et al.(1994), which was based on the x-ray crystal
structure of bFGF (Zhang et al., 1991; Zhu et al.,
1991; Eriksson et al., 1991). The crystallographically
determined structure of a protein represents its static state, in which
packing forces may play a major role in determining some of the
conformational features. Therefore, in our work we considered it
desirable to derive a solvent-immersed protein structure by
computationally solvating the crystallographic bFGF structure in a bath
of water and performing a molecular dynamics simulation. Structures
from the dynamics trajectory were analyzed for conformational features,
particularly at the putative receptor binding site. Solvent
accessibility calculations were then carried out at the putative
receptor binding site. The solvent accessible residues at positions
107, 109-114, and 96 were mutated to alanine or other residues.
The resulting muteins were purified to near homogeneity and then
compared with wild type bFGF for their receptor binding affinities to
soluble FGFR1-TPA fusion protein.
The molecular dynamics simulation was carried out at 300 K and 1 atm pressure. After 50 ps of equilibration, data were collected every picosecond over a period of 500 ps. Each conformer obtained at 25-ps intervals was minimized and stored for further analysis. The computations were performed using an extensively modified version (Ramnarayan et al., 1990) of the AMBER program (Singh et al., 1986). The time-averaged conformations resulting from the molecular dynamics calculations were analyzed to derive information for the site-directed mutagenesis studies.
These site mutagenesis studies were initiated to identify the
critical region on bFGF for the high affinity receptor binding. Peptide
mapping studies (Baird et al., 1988) have shown that a peptide
related to residues 106-115 of bFGF can inhibit the binding of I-bFGF to its receptor. Subsequently, the
three-dimensional structure determination of bFGF (Zhang et
al., 1991; Eriksson et al., 1993) suggested that these
residues form an antiparallel
-turn on the surface of the
molecule. To identify residues on bFGF for the receptor binding, the
crystal structure is first subjected to molecular dynamics treatment.
The root mean square deviation of the crystal structure, and the
molecular dynamics refined structure is then computed to be 2.01
Å for the
-carbon trace, 2.67 Å for the backbone
atoms, and 2.98 Å for all atoms in the system, indicating that
the solution structure of the protein is relatively close to the
crystal structure. Since the molecular dynamics refined structure of
bFGF is a more accurate presentation of the solution structure of the
protein, the water-accessible surface area (in Å
) of
each residue reported here is calculated based on the solvent
accessibility over the complete simulation period.
Figure 1:
A linear representation of a partial
structure of bFGF illustrating the mutations examined in this
investigation. The strands are represented by bars;
their starts and stops are indicated. The loops connecting the strands
are represented by straight lines.
Figure 4: Alignment of the amino acid sequences of nine members of FGF family around residue 90-106 of bFGF. The FGF sequence was obtained from protein data bank. The alignment of the primary amino acid sequence was done using the Genalign program: aFGF, FGF1; bFGF, FGF2; KGF, FGF7. Amino acid residues for Glu-96 and Tyr-106 are shown in boldface type.
Figure 3:
A close-up view of hydrogen bonding
between the side chains of Glu-96 and Tyr-106. The amino acid side
chains for Glu-96 and Tyr-106 are displayed in red and gold, respectively, the hydrogen bond is displayed in green, and the remaining residues are shown in the ribbon
diagram of the C backbone of
bFGF.
Several attempts have been made to define residues that participate in the FGF-FGFR interactions. An early peptide mapping approach by Baird et al.(1988) suggested that residues 106-115 are involved in receptor binding. Later, studies of the three-dimensional structure of bFGF revealed that part of this putative receptor binding peptide forms a loop consisting of residues 109-114 on the surface of the molecule (Eriksson et al., 1993). In addition, Seno et al.(1990) studied the action of truncated N- and C-terminal forms of bFGF on mitogenesis and heparin binding. Their results indicate that an essential part of bFGF for receptor binding is present within the sequence Asp-41 to Ser-100.
Recently, Pantoliano et al.(1994) described multivalent
ligand-receptor binding interactions in the fibroblast growth factor
system based on isothermal titration calorimetry and molecular
modeling. Their results indicate that the KYTSW
loop is a low affinity binding site required for receptor
dimerization in vitro and mitogenic signal transduction in
vivo. In a subsequent publication, Springer et al.(1994)
defined the primary high affinity FGFR binding site as comprising the
four hydrophobic amino acids (Tyr-24, Tyr-103, Leu-140, and Met-142)
and the two polar residues (Arg-44 and Asn-101).
In our work, we
employed site-directed mutagenesis based on the dynamic structure of
bFGF to determine which region is responsible for high affinity binding
to FGFR1. Substitution of residues at positions 107 and
109-114 by alanine or phenylalanine gave muteins with similar
receptor binding affinities compared with wild type bFGF. To extend
these studies, we identified the neighboring side chain Glu-96, which
is solvent accessible and conserved among the nine member of FGF family
( Fig. 4and Table 2). Upon replacement of Glu-96 with
alanine, a 1000-fold decrease in receptor binding was observed. This
result suggests that the conserved glutamic acid residue in other
members of FGF family may also play an important role in high affinity
receptor binding. To address the question of whether the drastic loss
of receptor binding affinity of E96A mutein is due to a global
conformational change, we considered the following evidence. First,
like wild type bFGF, E96A binds tightly to heparin-Sepharose, which is
the hallmark of specific interaction between bFGF and heparin (Thompson et al., 1994). Second, the E96A mutein can be precipitated
with specific monoclonal antibodies that recognize only the native form
of bFGF (data not shown). Finally, molecular dynamics studies revealed
no global change for the E96A mutein (data not shown).
To elucidate
the nature of the interaction between Glu-96 and FGFR1, the
anionic Glu-96 was changed to neutral glutamine. The resulting mutein
showed a 10-fold reduction in receptor binding, suggesting that
electrostatic interaction is involved in the receptor binding.
Furthermore, substitution of Glu-96 with a cationic residue, lysine,
gave more than a 300-fold reduction of receptor binding, providing
further evidence that electrostatic interaction in this region is an
important component of receptor binding.
To investigate whether the internal hydrogen bond between Glu-96 and the phenolic hydroxyl in Tyr-106 seen in our energy-refined structure of bFGF (Fig. 3) is important for the high affinity receptor binding of bFGF, we replaced Tyr-106 with phenylalanine. The resulting mutein, Y106F, had an almost 5-fold reduction of receptor binding affinity, suggesting that an internal hydrogen bond contributes to the receptor binding affinity.
Two likely mechanisms by which the loss of this hydrogen bond could negatively affect receptor binding were evaluated. Although destabilization of the protein structure of a bFGF mutein which lacks a buried hydrogen bond between Glu-96 and Tyr-106 might be expected, the behavior of the corresponding Y106F mutein, in which this hydrogen bond is also absent, does not support this possibility. Y106F mutein is a stable molecule that can be overexpressed at 37 °C and like wild type bFGF, binds tightly to a heparin-Sepharose column at 22 °C. Moreover, Y106F has only a 5-fold reduction in receptor binding, indicating that the dramatic loss of receptor binding activity of E96A is due principally to the loss of the carboxylate group of Glu-96, rather than to the loss of the buried hydrogen bond.
It is more probable that the Y106F mutein possesses a higher entropic state in comparison with wild type bFGF, owing to the gain in the freedom of the rotation of the carboxylate group of Glu-96 in the absence of hydrogen bond formation with the phenolic hydroxyl group of Tyr-106. As has been pointed out (Somers et al., 1994; Kossiakoff et al., 1994), the entropic states of the unbound partners are of importance in other hormone-receptor protein-protein interactions. In the present instance, by contrast to the Y106F mutein, the lower entropic state associated with restricted rotation due to hydrogen bonding is already present in unbound wild type bFGF, and a correspondingly smaller entropy loss, with consequent greater affinity, is therefore resulting from receptor binding.
Recently, Ornitz et al.(1995) described the crystal structure studies of the complex between bFGF and a synthetic trisaccharide. On the basis of their results, these workers suggested that a pair of trisaccharide binding sites derived from basic amino acid residues on bFGF (sites 2 and 2`) may be involved in the oligomerization of bFGF. In consideration of the experimentally determined crystal structure of the complex, site 2 is formed by Glu-96, Arg-97, and Arg-60, whereas site 2` comprises Arg-72, Lys-77, Arg-81, and Lys-86. According to these authors, site 2` may have a higher affinity for the trisaccharide than site 2, owing to shorter hydrogen bond lengths in site 2` relative to site 2. Since this crystal structure differs considerably from the arrangement postulated by Pantoliano et al.(1994) for the bioactive complex between heparin and bFGF, it is pertinent to inquire which of the two our present results support.
We note first that the structures of the
bFGF-oligosaccharide complexes obtained experimentally by Ornitz et
al.(1995) may not reflect the structure of the putative bioactive
complex. Moreover, we conclude for several reasons that the drastic
decrease we observe in the binding of the E96A mutein to FGFR1 cannot
be attributed to the impaired heparin binding, as would be required by
the experimental structure of Ornitz et al.(1995). First,
computer modeling ()indicates that substitution of Glu-96
with lysine does not appear to interfere with the hydrogen bond
formation between the conformationally mobile side chain of Lys and a
negatively charged trisaccharide or heparin moiety. This is at variance
with the nearly 300-fold reduction we found for the receptor binding of
the E96K mutein compared with wild type bFGF (Table 2), which
cannot therefore readily be explained on the basis of the impaired
heparin binding.
A similar situation exists in the case of Arg-97, which forms a hydrogen bond with the trisaccharide in the Ornitz structure. On the one hand, we found that replacement of Arg-97 with alanine, which is incapable of forming such a hydrogen bond, gives the mutein R97A, which binds tightly to the heparin-Sepharose and requires 25 mM Tris buffer containing 2 M NaCl for elution. On the other hand the mutein R97A has nearly undiminished affinity for the receptor (Table 2). These data argue against the possibility that the Ornitz structure is representative of the bioactive complex.
Last, FGF receptor dimerization is thought to proceed by a similar molecular mechanism for both bFGF and aFGF. Alignment of amino acid sequences in aFGF and bFGF reveals that the residues comprising the heparin binding site 2` on bFGF correspond to Gln-63, Asp-68, Leu-72, and Gln-77 on aFGF, none of which are either basic or conserved residues (Eriksson et al., 1993). This argues against the possibility that these particular residues on aFGF interact with trisaccharide or heparin and likewise makes it difficult to reconcile the Ornitz model with the induction of FGF receptor dimerization by aFGF.
The site-directed mutagenesis studies in this paper are
consistent with the current view that region 106-115 is not the
high affinity receptor binding site (Springer et al., 1994).
In addition, we have identified Glu-96 as a newly discovered crucial
residue for binding to FGFR1 by a electrostatic interaction that
is not predicted by the previous model (Pantoliano et al.,
1995). If we include Glu-96 as a extended high affinity site, the
surface area becomes 520 Å
. Since Glu-96 is situated
approximately between high and low affinity sites, this may indicate
that other residues between Glu-96 and the high affinity site are
involved in the high affinity binding. Currently, we are examining
residues in the neighbors of Glu-96.
Our results presented here do not support the possibility that the crystal complex between bFGF and trisaccharides examined by Ornitz is representative of a bioactive complex. These studies will aid in the understanding of the molecular interactions between ligand and receptor that is critical for the structure-based design of small molecule antagonists.