Proc. Nati. Acad. Sci. USA Vol. 88, pp. 3441-3445, April 1991 Biochemistry
Three-dimensional structure of human basic fibroblast growth factor (interleukin 1f/receptor/heparin binding site)
A. ELISABETH ERIKSSON*, LAWRENCE S. COUSENSt, LARRY H. WEAVER*,
BRIAN W. MATTHEWS* *Institute of Molecular Biology, Department of Physics, and Howard Hughes Medical Institute, University of Oregon, Eugene, OR 97403; and tChiron Corp. AND
and Protos Corp., 4560 Horton Street, Emeryville, CA 94608-2916
Contributed by Brian W. Matthews, December 28, 1990
ABSTRACT The three-dimensional structure of human basic fibroblast growth factor (bFGF) has been determined by x-ray crystallography and refined to a crystallographic residual of 17.4% at 2.2-A resolution. The structure was initially solved at a nominal resolution of 2.8 A by multiple isomorphous replacement using three heavy-atom derivatives. Although the map clearly showed the overall fold of the molecule, electron density was not observed for the first 19 amino-terminal and the last 3 carboxyl-terminal amino acids, suggesting that they are disordered. The bFGF crystals were grown from 2.0 M ammonium sulfate at pH 8.1 in space group P1 with cell dimnsions a = 30.9 A, b = 33.4 A, c = 35.9 A, a = 59.5, A = 72.00, and y = 75.60. There is one molecule per unit cell and the crystals diffract to spangs beyond 1.9 A. The overall strure of bFGF can be described as a trigonal pyramid with a fold very similar to that reported for interleukin 1.8, intereukin la, and soybean trypsin inhibitor. An apparent sulfate ion is bound within a basic region on the surface of the molecule and has as ligands the main-chain amide of Arg-120 and the side chains of Asn-27, Arg-120, and Lys-125. This is suggested as the presumed binding site for heparin. Residues 106-115, which are presumed to bind to the bFGF receptor [Baird, A., Schubert, D., Ling, N. & Guillemin, R. (1988) Proc. Nat!. Acad. Sci. USA 85, 2324-2328J, indude an irregular loop that extends somewhat from the surface of the protein and is about 25 A from the presumed heparin binding site. The backbone structure of this putative receptor-bnding loop is very similar, although not identical, to the correponding region of interleukin 1(3.
Basic fibroblast growth factor (bFGF) is a mitogenic, neurotrophic, and angiogenic polypeptide (1-3) that is a member of a family that now comprises seven related proteins (1, 4, 5), interacting with three related but distinct receptors (6, 7, 55). Several members of the FGF family are oncogenes (8-10), and the angiogenic properties of acidic FGF (aFGF) and bFGF (3) suggest several possible roles in tumor growth. bFGF also has wound-healing properties that have made it an attractive candidate as a therapeutic drug (11, 12). In addition, bFGF and an FGF receptor have been identified in Xenopus, where FGF is a potent inducer of mesoderm formation in developing embryos (13, 14). The diversity within the FGF family, including their receptors, and the many biological activities associated with these molecules may reflect complex mechanisms of cell differentiation and growth control. To develop an understanding of the interactions between the various members of the FGF family and the receptors at a molecular level, we have determined the three-dimensional structure of bFGFA The structure is very similar to that of interleukin 1,8 (IL-1,B), which is distantly related to the FGF family on the basis of primary sequence (15). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
3441
Structure Determination
Human bFGF was produced in genetically engineered yeast and purified by heparin-Sepharose affinity chromatography (16-18). Triclinic crystals (space group P1 with unit cell parameters a = 30.9 A, b = 33.4 A, c = 35.9 A, a = 59.5°, f3 = 72.00, and y = 75.60) were obtained by vapor diffusion methods. Five microliters of bFGF (15 mg/ml) was mixed on a coverslip with 5 1.l of well solution, typically 2.0 M (NH4)2SO4/0.1 M NaCl/0.1 M Tris HCl, pH 8.1/0.1% (vol/ vol) 2-mercaptoethanol, and equilibrated at room temperature over 1 ml ofwell solution in a Linbro tissue culture plate. With this method some small crystals suitable for data collection were obtained within a week. The reproducibility and size of the crystals were significantly improved by macroseedift similar to the procedure described by Thaller et al. (19). Small crystals of bFGF were washed in the above-described well solution. After at least 2 hr the solution was replaced, decreasing the (NH4)2SO4 concentration to 1.6 M. The crystals were left in this solution for 2-4 hr. After washing, each crystal was transferred to a sitting-drop tray containing S ,ul of bFGF and 5 ttl of the original well solution. The crystal was then allowed to grow over 0.5 ml of well solution. With this method crystal dimensions could be increased from 0.05 x 0.05 x 0.05 mm up to 0.2 x 0.2 x 0.4 mm within 1 week. High-resolution diffraction data sets for native and three derivative crystals were collected on a Xuong-Hamlin area detector (20) using graphite-monochromated CuKa radiation from a Rigaku RU200 rotating-anode generator. The positions of the heavy atoms (Table 1) were refined using the program HEAVY (21). The mean figure of merit, including anomalous scattering data in the phase determination, was 0.76 to 2.8-A resolution. A multiple isomorphous replacement (MIR) electrondensity map (Fig. 1) was calculated to 2.8-A resolution by using phases from all three derivatives, including their anomalous signal. The quality of the map was excellent and a model of the bFGF structure was built into the electron density by using the program FRODO (22). No density, however, was observed for the first 19 amino acids, suggesting that these residues are highly mobile or disordered. The MIR electron density was also weak for one loop region including residues 86-92, and these residues were therefore not included in the initial model. Refinement using the TNT least-squares refinement program package (23) reduced the R factor to 17.4% for 5126 reflections from 20.0- to 2.2-A resolution. The present model includes 24 water molecules and a bound sulfate ion but does not include residues 1-19, 87-89, and 144-146. The root Abbreviations: FGF, fibroblast growth factor; aFGF, acidic FGF; bFGF, basic FGF; IL-1, interleukin 1; MIR, multiple isomorphous replacement. tThe atomic coordinates have been deposited in the Protein Data Bank, Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973 (reference 1FGF).
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Biochemistry: Eriksson et al.
Proc. Nati. Acad. Sci. USA 88 (1991)
Table 1. Heavy-atom parameters
Phasing y z Derivative Occupancy x Ligand power 1.1 13.1 0.671 0.322 0.683 Cys-25 PCMBS 2.1 21.2 0.000 0.000 0.000 Cys-92 Hg(OAc)2 31.5 0.663 0.398 0.700 Cys-25 1.4 Cis-Pt 17.5 0.332 0.658 0.764 Met-142 PCMBS is p-chloromercuribenzenesulfonic acid (3-day soak, 2.0 mM), Hg(OAc)2 is mercuric acetate (5-day soak, 5.0 mM), and Cis-Pt is cis-diamminedichloroplatinum(II) (4-day soak, 5.0 mM). The occupancy is in arbitrary units. x, y, and z are in fractional coordinates with the origin arbitrarily assigned to coincide with the first binding site in the mercury derivative. The crystallographic thermal factorfor all sites was fixed as B = 30 A2. The phasing power, calculated to 2.8-A resolution, is defined as (FH)/E, where (FH) is the rms heavy-atom scattering and E is the rms lack of closure of the phase triangles.
-60 -XX-I---,-.--,,-120
xxI
mean square (rms) deviations of bond lengths and angles from ideal values are 0.016 A and 3.3°, respectively. The distribution of 4 and i angles is shown in Fig. 2. Structure of bFGF and Its Relation to IL-1u The backbone structure of bFGF (Fig. 3) can be described as a trigonal pyramid where the three sides are built of two p-strands together forming a 1-sheet barrel of six antiparallel strands. The base of the pyramid is built of six additional ,p-strands extending from the three sides of the pyramid to close one end of the barrel. Thus a threefold repeat is observed in the folding of the polypeptide chain and a pseudothreefold axis passes through the center of the base of the molecule and extends through the apex of the pyramid. Two buried water molecules and the side chain of Tyr-124 form hydrogen bonds that bridge between the three strands at the base of the molecule (see Fig. 3). There are also local threefold axes within each of the three sides of the pyramid. A buried water molecule is situated on each ofthese three local axes and forms hydrogen bonds to the threefold-related strands that come together around each threefold axis (Fig. 3). A weak correspondence has been noted between the amino acid sequences of aFGF (15), bFGF (25), and IL-1 (15, 26). Inspection of the overall fold ofbFGF immediately suggested that it is indeed similar to the known structure of IL-1i (27-29) as well as to IL-la (30, 31) and soybean trypsin inhibitor (32, 33). The backbone (Ce) coordinates of recombinant human IL-1i (28) were compared with those of bFGF by a method similar to that of Rossmann and Argos (34). In this procedure the two molecules are rotated and translated as rigid units so as to match as many Ca atoms as possible. Regions where the two structures differ substantially or have unequal numbers of CO atoms are excluded from the comparison. Fifty Cc atoms of IL-1i and bFGF could be superimposed with a rms discrepancy of 0.52 A. These occur within 9 of the 12 extended p-strands that comprise the core of bFGF, as well as the loop connecting the 9th and 10th strands (Fig. 4). The alignment of the amino acid sequences of bFGF and IL-1,B that corresponds to their structural superposition is
-180
-120
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0, degreeS FIG. 2. Ramachandran diagram (24) showing the distribution of and 4i angles for the refined model of bFGF. Glycine residues are indicated by squares and non-glycines by crosses. shown in Fig. 5. From residue 90 of bFGF to the carboxyl terminus this structure-based amino acid sequence alignment agrees within one amino acid with that proposed previously (1S). From the amino terminus to residue 89, however, the sequence alignment suggested previously is out of register by at least seven amino acids relative to that suggested by the structural correspondence. Thus, even though the initially proposed sequence alignment was based on a combination of methods and was essentially correct for about 40%o of bFGF and IL-1,B, it was also misleading for the first 601% of the respective molecules. It highlights the well-known difficulty of reliably aligning amino acid sequences when the sequence identity is weak (37). The 50 residues of bFGF and IL-1,8 whose a-carbons superimpose well in three dimensions (Fig. 4a) are marked in Fig. 5. Nine of these amino acids (18%) are identical. Overall, for the sequence alignment of bFGF and IL-1P shown in Fig. 5, the sequence identity is 12%. The amino acid sequence of aFGF is highly homologous (=53% sequence identity) with that of bFGF, suggesting that the two molecules have similar three-dimensional structures (15, 25). The present work further supports this idea. For the 50 residues whose a-carbons superimpose in bFGF and IL-1,8, the sequence identity between aFGF and bFGF is 68%o, suggesting that this part of the structure of aFGF is very similar to that observed in bFGF and IL-1,B. aFGF does, however, have an insertion of two amino acids in the surface loop (residues 109-114 of bFGF) that structurally corresponds in bFGF and IL-l,l3 (Fig. 5). There are, to date, seven members of the FGF family of peptides (1, 4, 5). Of the amino acids that are conserved within the family (e.g., see figure 1 of ref. 1), most are located within the core O-strand regions of bFGF, supporting the
i*
FIG. 1. MIR electron-density map in the vicinity of the presumed sulfate binding site, superimposed on the refined structural model. Oxygen atoms are drawn solid, carbon and sulfur open, and nitrogens are partially shaded. Contours drawn at a level of lo, where a- is the rms density throughout the unit cell.
Biochemistry: Eriksson et al.
E4~
Proc. Natl. Acad. Sci. USA 88 (1991)
3443
E4~ S143>*\\
SI43 P20
\K86
C69
P20
K86
C69
FIG. 3. Stereo drawing showing the overall structure of bFGF. The a-carbon backbone as seen in the electron density map begins at Pro-20 (P20) and ends at Ser-143 (S143). Residues 87-89 have weak, ill-defined electron density and are omitted from the figure. The direction of view is essentially parallel to an axis of approximate threefold symmetry. The isolated solid circles correspond to bound water molecules (see text). Also shown is the apparent bound sulfate ion (S04) and its ligands.
expectation that each of these proteins has an overall fold similar to that seen in bFGF. Possible Binding Site for Heparin Heparin is a highly sulfated glycosaminoglycan that has been shown to protect both basic and acidic FGF from acid and heat inactivation (38) as well as from proteolytic digestion (39-42). Heparin has also been shown to modify the activity of FGFs (43, 44) and to increase the binding affinity of aFGF for FGF receptors (45).
On the surface of bFGF there is a cluster of basic residues including Lys-26, Arg-44, Lys-119, Arg-120, Lys-125, Lys129, and Lys-135. In the MIR electron map an electron-dense feature was seen in this region that appeared to correspond to a bound sulfate ion (Fig. 1). The structure refinement supports this interpretation. The four sulfate oxygens are hydrogen-bonded by the side chains of Asn-27, Arg-120, and Lys-125 as well as the main-chain amide of Arg-120. Lys-119 is also close (Fig. 1). These data suggest that this is the binding site for the sulfate group of heparin. The residues that
a
D90
FIG. 4. (a) Superposition of the parts of the a-carbon backbones of bFGF (solid bonds) and IL-1P (open bonds) that most closely correspond. The numbering is for bFGF. (b) Superposition of the complete a-carbon backbone of bFGF (solid bonds) on that of IL-1,8 (open bonds). The sequence numbering is for bFGF except for (I)A117 and (I)S269, which indicate, respectively, Ala-117 and Ser-269, the amino-terminal and carboxyl-terminal residues in the crystal structure of IL-1,8 (27, 28).
3444
Biochemistry: Eriksson et al.
Proc. Natl. Acad Sci. USA 88 (1991) *
bFGF aFGF
IL-1
1 PRO ALA 1 117
LEU
*a *a *a *a
PRO GLU ASP GLY GLY SER GLY ALA PHE PROPRO GLYHIS PHE j7]ASP PRO LYS ARG 1 PHE ASN LEU PRO LEU GASN TYR LYS PRO LYS LEU 117 ALA PRO VAL ARG SER LEU ASN CYS THR
ASN GLY GLY LEU TYR CYS LEU TYR CYS SER ASN GLY GLY ARG
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30 PHE HE LEU ARG ILE HIS PRO ASP GLY ARG VAL ASP GLY VAL ARG GLU LYS SER ASP PRO HIS ILE --- --- LYS GLN LEU GLN 21 TYR PHE LEU ARG ILE LEU PRO ASP GLY THR VAL ASP GLY THR LYS ASP ARG [ER ASP GLN HIS ILE --GLN LEU GLN LEU CYS 133 SER LEU VAL MET SER GLY PRO TYR GLU LEU LYS ALA LEU HIS LEU GLN GLY GLN ASP MET GLU GLN GLN VAL VAL PHE SER MET SER ---
*0 0
0
*0 *0 *0 *0 *0 0
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57 ALA GLU GLU --bFGF ARG VAL SER ILE LYS GLY VAL CYS ALAE JARG LEU ALA MET LYS GLU 48 ALA GLU SER --aFGF ILE L9JGLU VAL TYR ILE LYS SER THR THR GLY GLN PHE LEUALA MET ASP THR jMJ ] LEU TYR LEUSER CYS VAL LEU LYS IL-la 162 PHE VAL GUN GLY GLU GLU SER ASN ASP LYS ILE PRO VAL ALA LEU GLY LEU LYS [ELYS ---
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* *0 bFGF 80 --ARG LEU LEU ALA LYS CYS VAL THR --ASP PHE CYS PHE --aFGF 71 --GLN THR PRO ASN --LEUILEU TYR GLY j GLU G CYS LEU PHE LEU G IL-la 191 ASP ASP LYS PRO THRLEU GUN LEU GLU SER VAL ASP PRO LYS ASN TYR PRO LYS LYS LYS MET GLU LYS ARG PHE VAL PHE ASN LYS ---
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97 ARG LEU GLU SER ASN ASN TYR ASN THR TYR ARG SERARG LY]TYR THR GLN SER TRP TYR VAL ALA LEU LYS ARG THR 88 ARG LEU GLU GLU ASH TYR ASN THR TYR ILE SERI LYS HIS ALA GLU LYS HIS TRP ARG iVAL GLY LEU LYSLYS ASN 220 ILE GLU ILE --- ASN ASN LYS LEU GLU PHE GLU SER ALA GLN PHE PRO --- --- ASN TRP TYR ILE SER THR SER GUI ALA GLU ASN
bFGF aFGF IL-1
146 GLY PRO --- GLY GUN LYS ALA ILE LEU PHE LEU PRO MET SER ALA LYS E 124 TYR YS LEU GLY SER LYS 117 SER LYS LEU GLY PRO ARG HIS PHE --- GLY GLN LYS ALA ILE LEU PHE LEU PRO LEU PRO J LSERSERASP 140 P 246 MET PRO VAL PHE LEU GLY GLY THR LYS GLY GLY THR MET GLN PHE VAL SER SER 269 ASP ILE THR A
---
aao a a a a)
---
* * *a*a*a GI
FIG. 5. Alignment of the amino acid sequences of human bFGF (35) [sequence numbered to correspond to bovine bFGF (25)] and IL-113 (15, 26) suggested by the correspondence between their three-dimensional crystal structures. The sequence of bovine aFGF (15) is also included. The 50 residues whose a-carbons correspond with a rms discrepancy of 0.52 A are indicated with asterisks. Residues in bFGF that are indicated by the algorithm of Kabsch and Sander (36) as having a 13-sheet conformation are indicated (13). There are 10 such "3-sheet strands" shown in the figure. There are two additional segments, comprising residues 117-119 and 124-129 and indicated (J . . . 13), that do not meet the Kabsch and Sander criteria for 13-sheet strands, but, nevertheless, form the 10th and 11th of the 12 18-strands that comprise the overall framework of the bFGF and IL-1,8 structures.
correspond to Asn-27, Arg-120, and Lys-125 in the amino acid sequence of aFGF (Fig. 5) are, respectively, Asn-18, Lys-113, and Lys-118. The methylation of Lys-118 of aFGF (46), or its replacement by a glutamic acid (1), significantly reduces the affinity of aFGF for immobilized heparin. It seems likely that Asn-27, Arg-120, and Lys-125 on bFGF and Asn-18, Lys-113, and Lys-118 on aFGF constitute binding sites for heparin and other sulfated substrates. In contrast, however, the three corresponding amino acids in IL-1,3 (Fig. 5) are Gln-120, Gln-242, and Pro-247, making it very unlikely that this could be a sulfate or heparin binding site on the interleukin structure. Electron density, possibly compatible with a second bound sulfate ion, was observed in the bFGF crystal structure about 8 A from the first site. The apparent hydrogen-bonding groups include the main-chain amide of Leu-126 and the side chains of Lys-119 and Lys-129. In this case, however, the electron density is not as strong and as clearly defined as at the first site. Cysteine Locations There are four cysteines in bFGF, located at positions 25, 69, 87, and 92. Cys-25 and Cys-92 are highly conserved in different members of the FGF family (1, 4, 5). Whether this or another pair of cysteines might form a disulfide bond has been the subject of debate (1, 47-49). All four cysteines have been replaced with serines by two independent groups but with somewhat different results. Seno et al. (47) observed a decrease in biological activity, whereas Arakawa et al. (49) reported no significant effect on FGF activity. In the crystal structure of bFGF the electron density defining the positions
of Cys-25, -69, and -92 is clear. Both Cys-69 and Cys-92 are exposed to solvent, whereas Cys-25 is completely buried in a loop region. It seems clear that there is no possibility of disulfide formation between these three well-defined cysteines. Cys-25 is situated 21 A from both Cys-69 and Cys-92, and the distance between Cys-69 and Cys-92 is 19 A. The only cysteines situated close enough to possibly form a disulfide bond are Cys-87 and Cys-92, and the electron density in the vicinity of Cys-87 is extremely diffuse. These two residues, however, have never been suggested to form such a bond (47-49). Receptor Binding
The peptide fragments 24-68 and 106-115 of bFGF bind to heparin, interact with the bFGF receptor, and have agonist activity in mitogenic assays for bFGF (42). The first peptide has relatively low potency but the second displays all the features expected of a receptor-binding peptide. Fragment 106-115 begins in the middle of the 9th /-strand, makes a somewhat open loop on the surface of the bFGF molecule, and terminates at the beginning of the 10th /-strand (Figs. 3 and 5). Fragment 106-115 is also within the most extended segment of bFGF that structurally corresponds to IL-1p (Figs. 4a and 5). At face value this structural conservation within the putative receptor-binding region might suggest that the backbone conformation is common to a broad family of receptor-binding proteins and that the differences in amino acid sequence within this conserved motif could account for the discrimination between different receptors. It would be attractive to consider that receptor recognition by a conserved "strand-turn-strand" motif in bFGF, IL-101, and other
Biochemistry: Eriksson et A putative factors might be a counterpart to the use of the conserved "helix-turn-helix" motif in the recognition of specific binding sites on DNA (50). Further inspection, however, suggests that the basis of recognition and discrimination in the case of bFGF and IL-1,/ may be more complicated. Within the solvent-exposed loop region, residues 111-114 of bFGF form a type I turn (51). In contrast, the corresponding residues in IL-1,3, 233-236, form a type II turn. This is a significant difference in the geometry of the respective peptide backbones. Perhaps more dramatic is the apparent difference in this region between aFGF and bFGF. aFGF and bFGF bind to similar receptors, but with different affinity for different types of receptor species (52). According to the sequence alignment shown in Fig. 5, however, aFGF has two additional amino acids inserted at or near the position corresponding to the 112-113 peptide bond of bFGF. Therefore the loop in aFGF that corresponds to the 106-115 fragment of bFGF cannot have the same backbone conformation. It could be that the parts of aFGF and bFGF that contact receptor do not specifically include the region of the dipeptide insertion, but this remains to be determined. The presumed receptor-binding region of bFGF is about 25 A from the presumed heparin binding site and the two sites are on different faces of the molecule (Fig. 3). This suggests that receptor binding and the binding of heparin are likely to be more-or-less independent, as is also indicated by the finding that neutralizing antibodies inhibit the binding of bFGF to its receptor but not to heparin (53). The apparent separation of the heparin and receptor binding sites also suggests that the potentiating effect of heparin on the mitogenic activity of aFGF and its inhibition of the growthpromoting activity of bFGF (42-44) are due to indirect effects. Bulky polyanionic compounds, such as suramin, that inhibit the receptor binding of both aFGF and bFGF (54) could do so by binding to FGF and preventing access to the receptor-binding region or by causing a conformational change in the FGF molecule. We are most grateful to Dr. Dale Tronrud, Dr. Jim Remington, and especially Dr. Steve Roderick for extensive help and advice with the structure determination. A.E.E. extends special thanks to Dr. Sherry Mowbray, Uppsala University, Sweden, for her suggestion of using macroseeding as a method for obtaining large beautiful crystals. We thank Drs. J. Priestle and M. Grutter, CIBA-Geigy, for supplying the refined coordinates of IL-1,t (also available from the Brookhaven Protein Data Bank). A.E.E. acknowledges the support of the Swed-
ish Natural Science Research Council. L.S.C. thanks Drs. Steve
Rosenberg and Pablo Valenzuela for their support and helpful advice. This work was supported in part by grants to B.W.M. from the National Institutes of Health (GM20066), the National Science Foundation (DMB8611084), and the Lucille P. Markey Charitable Trust. 1. Burgess, W. H. & Maciag, T. (1989) Annu. Rev. Biochem. 58, 575-606. 2. Walicke, P., Cowan, M. W., Ueno, N., Baird, A. & Guillemin, R. (1986) Proc. Nati. Acad. Sci. USA 83, 3012-3016. 3. Folkman, J. & Klagsbrun, M. (1987) Science 235, 442-447. 4. Marics, I., Adelaide, J., Raybaud, F., Mattei, M. G., Coulier, F., Planche, J., de Lapeyriere, 0. & Birnbaum, D. (1989) Oncogene 4, 335-340. 5. Finch, P. W., Rubin, J. S., Miki, T., Ron, D. & Aaronson, S. A. (1989) Science 245, 752-755. 6. Lee, P. L., Johnson, D. E., Cousens, L. S., Fried, V. A. & Williams, L. T. (1989) Science 245, 57-60. 7. Dionne, C. A., Crumley, G., Bellot, F., Kaplow, J. M., Searfoss, G., Ruta, M., Burgess, W. H., Jaye, M. & Schlessinger, J. (1990) EMBO J. 9, 2685-2692. 8. Dickson, C. & Peters, G. (1987) Nature (London) 326, 833 (lett.). 9. Delli-Bovi, P., Curatola, A. M., Kern, F., Greco, A., Ittman, M. & Basilico, C. (1987) Cell 50, 729-737.
Proc. Natl. Acad. Sci. USA 88 (1991)
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10. Zhan, X., Bates, B., Hu, X. & Goldfarb, M. (1988) Mol. Cell. Biol. 8, 3487-3495. 11. ten Dijke, P. & Iwata, K. K. (1989) BiolTechnology 7, 793-798. 12. Tsubi, R. & Rifkin, D. B. (1990) J. Exp. Med. 172, 245-251. 13. Kimelman, D., Abraham, J. A., Haaparanta, T., Palisi, T. M. & Kirschner, M. (1989) Science 242, 1053-1056. 14. Musci, T. J., Amaya, E. & Kirschner, M. W. (1990) Proc. Natl. Acad. Sci. USA 87, 8365-8369. 15. Gimenez-Gallego, G., Rodkey, J., Bennett, C., Rios-Candelore, M., DiSalvo, J. & Thomas, K. (1985) Science 230, 1385-1388. 16. Barr, P. J., Cousens, L. S., Lee-Ng, C. T., Medina-Selby, A., Masiarz, F. R., Hallewell, R. A., Chamberlain, S. H., Bradley, J. D., Lee, D., Steimer, K. S., Poulter, L., Burlingame, A. L., Esch, F. & Baird, A. (1988) J. Biol. Chem. 263, 16471-16478. 17. Zhang, J., Cousens, L. S., Barr, P. J. & Sprang, S. R. (1991) Proc. Natl. Acad. Sdi. USA 88, 3446-3450. 18. Shing, Y., Folkman, J., Sullivan, R., Butterfield, C., Murray, J. & Klagsbrun, M. (1984) Science 223, 1296-1299. 19. Thaller, C., Weaver, L. H., Eichele, G., Wilson, E., Karlsson, R. & Jansonius, J. N. (1981) J. Mol. Biol. 147, 465-469. 20. Xuong, N. H., Nielsen, C., Hamlin, R. & Anderson, D. (1985) J. Appl. Crystallogr. 181, 342-350. 21. Terwilliger, T. C. & Eisenberg, D. (1983) Acta Crystallogr. Sect. A: Found. Crystallogr. 39, 813-817. 22. Jones, T. A. (1982) in Computational Crystallography, ed. Sayre, D. (Clarendon, Oxford), pp. 303-317. 23. Tronrud, D. E., Ten Eyck, L. F. & Matthews, B. W. (1987) Acta Crystallogr. Sect. A: Found. Crystallogr. 43, 489-503. 24. Ramachandran, G. N., Ramakrishnan, C. & Sasisekharan, V. (1963) J. Mol. Biol. 7, 95-99. 25. Esch, F., Baird, A., Ling, N., Ueno, N., Hill, F., Denoroy, L., Klepper, R., Gospodarowicz, D., Bohlen, P. & Guillemin, R. (1985) Proc. Natl. Acad. Sci. USA 82, 6507-6511. 26. Thomas, K. A. & Gimenez-Gallego, G. (1986) Trends Biochem. Sci. 11, 81-84. 27. Priestle, J. P., Schar, H.-P. & Grutter, M. G. (1988) EMBO J. 7, 339-343. 28. Priestle, J. P., Schar, H.-P. & Grutter, M. G. (1989) Proc. Natl. Acad. Sci. USA 86, 9667-9671. 29. Finzel, B. C., Clancy, L. L., Holland, D. R., Muchmore, S. W., Watenpaugh, K. D. & Einspahr, H. M. (1989) J. Mol. Biol. 209, 779-791. 30. Graves, B. J., Hatada, M. H., Hendrickson, W. A., Miller, J. K., Madison, V. S. & Satow, Y. (1990) Biochemistry 29, 2679-2684. 31. Einspahr, H. M., Clancy, L. L., Holland, D. R., Muchmore, S. W., Watenpaugh, K. D. & Finzel, B. C. (1990) in Current Research in Protein Chemistry, ed. Villafranca, J. J. (Academic, New York), pp. 351-358. 32. Sweet, R. M., Wright, H. T., Janin, J., Chothia, C. H. & Blow, D. M. (1974) Biochemistry 13, 4212-4228. 33. McLachlan, A. D. (1979) J. Mol. Biol. 133, 557-563. 34. Rossmann, M. G. & Argos, P. (1975) J. Biol. Chem. 250, 7525-7532. 35. Abraham, J. A., Whong, J. L., Tumolo, A., Mergia, A., Friedman, J., Gospodarowicz, D. & Fiddes, J. C. (1986) EMBO J. 5, 2523-2528. 36. Kabsch, W. & Sander, C. (1983) Biopolymers 22, 2577-2637. 37. James, M. N. G., Delbaere, L. T. J. & Brayer, G. D. (1978) Can. J. Biochem. 56, 396-402. 38. Gospodarowicz, D. & Cheng, J. (1986) J. Cell. Physiol. 128, 475-484. 39. Saksela, O., Moscatelli, D., Sommer, A. & Rifkin, D. B. (1988) J. Cell. Biol. 107, 743-751. 40. Damon, D. H., Lobb, R. R., D'Amore, P. A. & Wagner, J. A. (1989) J. Cell. Physiol. 138, 221-226. 41. Rosengart, T. K., Johnson, W. V., Friesel, R., Clark, R. & Maciag, T. (1988) Biochem. Biophys. Res. Commun. 152, 432-440. 42. Baird, A., Schubert, D., Ling, N. & Guillemin, R. (1988) Proc. Natl. Acad. Sci. USA 85, 2324-2328. 43. Neufeld, G., Gospodarowicz, D., Dodge, L. & Fujii, D. K. (1987) J. Cell. Physiol. 131, 131-140. 44. Ulrich, S., Lagente, O., Lenfant, M. & Courtois, Y. (1986) Biochem. Biophys. Res. Commun. 137, 1205-1213. 45. Kaplow, J. M., Bellot, F., Crumley, G., Dionne, C. A. & Jaye, M. (1990) Biochem. Biophys. Res. Commun. 172, 107-112. 46. Harper, J. W. & Lobb, R. R. (1988) Biochemistry 27, 671-678. 47. Seno, M., Sasada, R., Iwane, M., Sudo, K., Kurokawa, T., Ito, K. & Igarashi, K. (1988) Biochem. Biophys. Res. Commun. 151, 701-708. 48. Fox, G. M., Schiffer, S. G., Rohde, M. F., Tsai, L. B., Banks, A. R. & Arakawa, T. A. (1988) J. Biol. Chem. 263, 18452-18458. 49. Arakawa, T., Hsu, Y.-R., Schiffer, S. G., Tsai, L. B., Curless, C. & Fox, G. M. (1989) Biochem. Biophys. Res. Commun. 161, 335-341. 50. Brennan, R. G. & Matthews, B. W. (1989) J. Biol. Chem. 264, 19031906. 51. Venkatachalam, C. M. (1968) Biopolymers 6, 1425-1436. 52. Neufeld, G. & Gospodarowicz, D. (1986) J. Biol. Chem. 261, 5631-5637. 53. Kurokawa, M., Doctrow, S. R. & Klagsbrun, M. (1989) J. Biol. Chem. 264, 7686-7691. 54. Huang, J. S., Huang, S. S. & Kuo, M.-D. (1986) J. Biol. Chem. 261, 11600-11607. 55. Pasquale, E. B. (1990) Proc. Natl. Acad. Sci. USA 87, 5812-5816.
Three-dimensional structure of human basic fibroblast growth factor (interleukin 1f/receptor/heparin binding site)
A. ELISABETH ERIKSSON*, LAWRENCE S. COUSENSt, LARRY H. WEAVER*,
BRIAN W. MATTHEWS* *Institute of Molecular Biology, Department of Physics, and Howard Hughes Medical Institute, University of Oregon, Eugene, OR 97403; and tChiron Corp. AND
and Protos Corp., 4560 Horton Street, Emeryville, CA 94608-2916
Contributed by Brian W. Matthews, December 28, 1990
ABSTRACT The three-dimensional structure of human basic fibroblast growth factor (bFGF) has been determined by x-ray crystallography and refined to a crystallographic residual of 17.4% at 2.2-A resolution. The structure was initially solved at a nominal resolution of 2.8 A by multiple isomorphous replacement using three heavy-atom derivatives. Although the map clearly showed the overall fold of the molecule, electron density was not observed for the first 19 amino-terminal and the last 3 carboxyl-terminal amino acids, suggesting that they are disordered. The bFGF crystals were grown from 2.0 M ammonium sulfate at pH 8.1 in space group P1 with cell dimnsions a = 30.9 A, b = 33.4 A, c = 35.9 A, a = 59.5, A = 72.00, and y = 75.60. There is one molecule per unit cell and the crystals diffract to spangs beyond 1.9 A. The overall strure of bFGF can be described as a trigonal pyramid with a fold very similar to that reported for interleukin 1.8, intereukin la, and soybean trypsin inhibitor. An apparent sulfate ion is bound within a basic region on the surface of the molecule and has as ligands the main-chain amide of Arg-120 and the side chains of Asn-27, Arg-120, and Lys-125. This is suggested as the presumed binding site for heparin. Residues 106-115, which are presumed to bind to the bFGF receptor [Baird, A., Schubert, D., Ling, N. & Guillemin, R. (1988) Proc. Nat!. Acad. Sci. USA 85, 2324-2328J, indude an irregular loop that extends somewhat from the surface of the protein and is about 25 A from the presumed heparin binding site. The backbone structure of this putative receptor-bnding loop is very similar, although not identical, to the correponding region of interleukin 1(3.
Basic fibroblast growth factor (bFGF) is a mitogenic, neurotrophic, and angiogenic polypeptide (1-3) that is a member of a family that now comprises seven related proteins (1, 4, 5), interacting with three related but distinct receptors (6, 7, 55). Several members of the FGF family are oncogenes (8-10), and the angiogenic properties of acidic FGF (aFGF) and bFGF (3) suggest several possible roles in tumor growth. bFGF also has wound-healing properties that have made it an attractive candidate as a therapeutic drug (11, 12). In addition, bFGF and an FGF receptor have been identified in Xenopus, where FGF is a potent inducer of mesoderm formation in developing embryos (13, 14). The diversity within the FGF family, including their receptors, and the many biological activities associated with these molecules may reflect complex mechanisms of cell differentiation and growth control. To develop an understanding of the interactions between the various members of the FGF family and the receptors at a molecular level, we have determined the three-dimensional structure of bFGFA The structure is very similar to that of interleukin 1,8 (IL-1,B), which is distantly related to the FGF family on the basis of primary sequence (15). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
3441
Structure Determination
Human bFGF was produced in genetically engineered yeast and purified by heparin-Sepharose affinity chromatography (16-18). Triclinic crystals (space group P1 with unit cell parameters a = 30.9 A, b = 33.4 A, c = 35.9 A, a = 59.5°, f3 = 72.00, and y = 75.60) were obtained by vapor diffusion methods. Five microliters of bFGF (15 mg/ml) was mixed on a coverslip with 5 1.l of well solution, typically 2.0 M (NH4)2SO4/0.1 M NaCl/0.1 M Tris HCl, pH 8.1/0.1% (vol/ vol) 2-mercaptoethanol, and equilibrated at room temperature over 1 ml ofwell solution in a Linbro tissue culture plate. With this method some small crystals suitable for data collection were obtained within a week. The reproducibility and size of the crystals were significantly improved by macroseedift similar to the procedure described by Thaller et al. (19). Small crystals of bFGF were washed in the above-described well solution. After at least 2 hr the solution was replaced, decreasing the (NH4)2SO4 concentration to 1.6 M. The crystals were left in this solution for 2-4 hr. After washing, each crystal was transferred to a sitting-drop tray containing S ,ul of bFGF and 5 ttl of the original well solution. The crystal was then allowed to grow over 0.5 ml of well solution. With this method crystal dimensions could be increased from 0.05 x 0.05 x 0.05 mm up to 0.2 x 0.2 x 0.4 mm within 1 week. High-resolution diffraction data sets for native and three derivative crystals were collected on a Xuong-Hamlin area detector (20) using graphite-monochromated CuKa radiation from a Rigaku RU200 rotating-anode generator. The positions of the heavy atoms (Table 1) were refined using the program HEAVY (21). The mean figure of merit, including anomalous scattering data in the phase determination, was 0.76 to 2.8-A resolution. A multiple isomorphous replacement (MIR) electrondensity map (Fig. 1) was calculated to 2.8-A resolution by using phases from all three derivatives, including their anomalous signal. The quality of the map was excellent and a model of the bFGF structure was built into the electron density by using the program FRODO (22). No density, however, was observed for the first 19 amino acids, suggesting that these residues are highly mobile or disordered. The MIR electron density was also weak for one loop region including residues 86-92, and these residues were therefore not included in the initial model. Refinement using the TNT least-squares refinement program package (23) reduced the R factor to 17.4% for 5126 reflections from 20.0- to 2.2-A resolution. The present model includes 24 water molecules and a bound sulfate ion but does not include residues 1-19, 87-89, and 144-146. The root Abbreviations: FGF, fibroblast growth factor; aFGF, acidic FGF; bFGF, basic FGF; IL-1, interleukin 1; MIR, multiple isomorphous replacement. tThe atomic coordinates have been deposited in the Protein Data Bank, Chemistry Department, Brookhaven National Laboratory, Upton, NY 11973 (reference 1FGF).
3442
Biochemistry: Eriksson et al.
Proc. Nati. Acad. Sci. USA 88 (1991)
Table 1. Heavy-atom parameters
Phasing y z Derivative Occupancy x Ligand power 1.1 13.1 0.671 0.322 0.683 Cys-25 PCMBS 2.1 21.2 0.000 0.000 0.000 Cys-92 Hg(OAc)2 31.5 0.663 0.398 0.700 Cys-25 1.4 Cis-Pt 17.5 0.332 0.658 0.764 Met-142 PCMBS is p-chloromercuribenzenesulfonic acid (3-day soak, 2.0 mM), Hg(OAc)2 is mercuric acetate (5-day soak, 5.0 mM), and Cis-Pt is cis-diamminedichloroplatinum(II) (4-day soak, 5.0 mM). The occupancy is in arbitrary units. x, y, and z are in fractional coordinates with the origin arbitrarily assigned to coincide with the first binding site in the mercury derivative. The crystallographic thermal factorfor all sites was fixed as B = 30 A2. The phasing power, calculated to 2.8-A resolution, is defined as (FH)/E, where (FH) is the rms heavy-atom scattering and E is the rms lack of closure of the phase triangles.
-60 -XX-I---,-.--,,-120
xxI
mean square (rms) deviations of bond lengths and angles from ideal values are 0.016 A and 3.3°, respectively. The distribution of 4 and i angles is shown in Fig. 2. Structure of bFGF and Its Relation to IL-1u The backbone structure of bFGF (Fig. 3) can be described as a trigonal pyramid where the three sides are built of two p-strands together forming a 1-sheet barrel of six antiparallel strands. The base of the pyramid is built of six additional ,p-strands extending from the three sides of the pyramid to close one end of the barrel. Thus a threefold repeat is observed in the folding of the polypeptide chain and a pseudothreefold axis passes through the center of the base of the molecule and extends through the apex of the pyramid. Two buried water molecules and the side chain of Tyr-124 form hydrogen bonds that bridge between the three strands at the base of the molecule (see Fig. 3). There are also local threefold axes within each of the three sides of the pyramid. A buried water molecule is situated on each ofthese three local axes and forms hydrogen bonds to the threefold-related strands that come together around each threefold axis (Fig. 3). A weak correspondence has been noted between the amino acid sequences of aFGF (15), bFGF (25), and IL-1 (15, 26). Inspection of the overall fold ofbFGF immediately suggested that it is indeed similar to the known structure of IL-1i (27-29) as well as to IL-la (30, 31) and soybean trypsin inhibitor (32, 33). The backbone (Ce) coordinates of recombinant human IL-1i (28) were compared with those of bFGF by a method similar to that of Rossmann and Argos (34). In this procedure the two molecules are rotated and translated as rigid units so as to match as many Ca atoms as possible. Regions where the two structures differ substantially or have unequal numbers of CO atoms are excluded from the comparison. Fifty Cc atoms of IL-1i and bFGF could be superimposed with a rms discrepancy of 0.52 A. These occur within 9 of the 12 extended p-strands that comprise the core of bFGF, as well as the loop connecting the 9th and 10th strands (Fig. 4). The alignment of the amino acid sequences of bFGF and IL-1,B that corresponds to their structural superposition is
-180
-120
I
-60
0
60
120
9 ¶4
-J
-
3
1*9
0
4tw ' 0
. .F.
0
* XX
180
0, degreeS FIG. 2. Ramachandran diagram (24) showing the distribution of and 4i angles for the refined model of bFGF. Glycine residues are indicated by squares and non-glycines by crosses. shown in Fig. 5. From residue 90 of bFGF to the carboxyl terminus this structure-based amino acid sequence alignment agrees within one amino acid with that proposed previously (1S). From the amino terminus to residue 89, however, the sequence alignment suggested previously is out of register by at least seven amino acids relative to that suggested by the structural correspondence. Thus, even though the initially proposed sequence alignment was based on a combination of methods and was essentially correct for about 40%o of bFGF and IL-1,B, it was also misleading for the first 601% of the respective molecules. It highlights the well-known difficulty of reliably aligning amino acid sequences when the sequence identity is weak (37). The 50 residues of bFGF and IL-1,8 whose a-carbons superimpose well in three dimensions (Fig. 4a) are marked in Fig. 5. Nine of these amino acids (18%) are identical. Overall, for the sequence alignment of bFGF and IL-1P shown in Fig. 5, the sequence identity is 12%. The amino acid sequence of aFGF is highly homologous (=53% sequence identity) with that of bFGF, suggesting that the two molecules have similar three-dimensional structures (15, 25). The present work further supports this idea. For the 50 residues whose a-carbons superimpose in bFGF and IL-1,8, the sequence identity between aFGF and bFGF is 68%o, suggesting that this part of the structure of aFGF is very similar to that observed in bFGF and IL-1,B. aFGF does, however, have an insertion of two amino acids in the surface loop (residues 109-114 of bFGF) that structurally corresponds in bFGF and IL-l,l3 (Fig. 5). There are, to date, seven members of the FGF family of peptides (1, 4, 5). Of the amino acids that are conserved within the family (e.g., see figure 1 of ref. 1), most are located within the core O-strand regions of bFGF, supporting the
i*
FIG. 1. MIR electron-density map in the vicinity of the presumed sulfate binding site, superimposed on the refined structural model. Oxygen atoms are drawn solid, carbon and sulfur open, and nitrogens are partially shaded. Contours drawn at a level of lo, where a- is the rms density throughout the unit cell.
Biochemistry: Eriksson et al.
E4~
Proc. Natl. Acad. Sci. USA 88 (1991)
3443
E4~ S143>*\\
SI43 P20
\K86
C69
P20
K86
C69
FIG. 3. Stereo drawing showing the overall structure of bFGF. The a-carbon backbone as seen in the electron density map begins at Pro-20 (P20) and ends at Ser-143 (S143). Residues 87-89 have weak, ill-defined electron density and are omitted from the figure. The direction of view is essentially parallel to an axis of approximate threefold symmetry. The isolated solid circles correspond to bound water molecules (see text). Also shown is the apparent bound sulfate ion (S04) and its ligands.
expectation that each of these proteins has an overall fold similar to that seen in bFGF. Possible Binding Site for Heparin Heparin is a highly sulfated glycosaminoglycan that has been shown to protect both basic and acidic FGF from acid and heat inactivation (38) as well as from proteolytic digestion (39-42). Heparin has also been shown to modify the activity of FGFs (43, 44) and to increase the binding affinity of aFGF for FGF receptors (45).
On the surface of bFGF there is a cluster of basic residues including Lys-26, Arg-44, Lys-119, Arg-120, Lys-125, Lys129, and Lys-135. In the MIR electron map an electron-dense feature was seen in this region that appeared to correspond to a bound sulfate ion (Fig. 1). The structure refinement supports this interpretation. The four sulfate oxygens are hydrogen-bonded by the side chains of Asn-27, Arg-120, and Lys-125 as well as the main-chain amide of Arg-120. Lys-119 is also close (Fig. 1). These data suggest that this is the binding site for the sulfate group of heparin. The residues that
a
D90
FIG. 4. (a) Superposition of the parts of the a-carbon backbones of bFGF (solid bonds) and IL-1P (open bonds) that most closely correspond. The numbering is for bFGF. (b) Superposition of the complete a-carbon backbone of bFGF (solid bonds) on that of IL-1,8 (open bonds). The sequence numbering is for bFGF except for (I)A117 and (I)S269, which indicate, respectively, Ala-117 and Ser-269, the amino-terminal and carboxyl-terminal residues in the crystal structure of IL-1,8 (27, 28).
3444
Biochemistry: Eriksson et al.
Proc. Natl. Acad Sci. USA 88 (1991) *
bFGF aFGF
IL-1
1 PRO ALA 1 117
LEU
*a *a *a *a
PRO GLU ASP GLY GLY SER GLY ALA PHE PROPRO GLYHIS PHE j7]ASP PRO LYS ARG 1 PHE ASN LEU PRO LEU GASN TYR LYS PRO LYS LEU 117 ALA PRO VAL ARG SER LEU ASN CYS THR
ASN GLY GLY LEU TYR CYS LEU TYR CYS SER ASN GLY GLY ARG
ASPISERJGLN *
bFGF aFGF
IL-1a
GLN LYS
*a *a *a *3
30 PHE HE LEU ARG ILE HIS PRO ASP GLY ARG VAL ASP GLY VAL ARG GLU LYS SER ASP PRO HIS ILE --- --- LYS GLN LEU GLN 21 TYR PHE LEU ARG ILE LEU PRO ASP GLY THR VAL ASP GLY THR LYS ASP ARG [ER ASP GLN HIS ILE --GLN LEU GLN LEU CYS 133 SER LEU VAL MET SER GLY PRO TYR GLU LEU LYS ALA LEU HIS LEU GLN GLY GLN ASP MET GLU GLN GLN VAL VAL PHE SER MET SER ---
*0 0
0
*0 *0 *0 *0 *0 0
0
*0 *0 *0 0
57 ALA GLU GLU --bFGF ARG VAL SER ILE LYS GLY VAL CYS ALAE JARG LEU ALA MET LYS GLU 48 ALA GLU SER --aFGF ILE L9JGLU VAL TYR ILE LYS SER THR THR GLY GLN PHE LEUALA MET ASP THR jMJ ] LEU TYR LEUSER CYS VAL LEU LYS IL-la 162 PHE VAL GUN GLY GLU GLU SER ASN ASP LYS ILE PRO VAL ALA LEU GLY LEU LYS [ELYS ---
---
---
---
---
---
---
---
---
---
a *a *a *a
*a *a
*a
* *0 bFGF 80 --ARG LEU LEU ALA LYS CYS VAL THR --ASP PHE CYS PHE --aFGF 71 --GLN THR PRO ASN --LEUILEU TYR GLY j GLU G CYS LEU PHE LEU G IL-la 191 ASP ASP LYS PRO THRLEU GUN LEU GLU SER VAL ASP PRO LYS ASN TYR PRO LYS LYS LYS MET GLU LYS ARG PHE VAL PHE ASN LYS ---
---
---
---
---
---
---
---
*
*
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---
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---
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*
*
bFGF aFGF IL-1
97 ARG LEU GLU SER ASN ASN TYR ASN THR TYR ARG SERARG LY]TYR THR GLN SER TRP TYR VAL ALA LEU LYS ARG THR 88 ARG LEU GLU GLU ASH TYR ASN THR TYR ILE SERI LYS HIS ALA GLU LYS HIS TRP ARG iVAL GLY LEU LYSLYS ASN 220 ILE GLU ILE --- ASN ASN LYS LEU GLU PHE GLU SER ALA GLN PHE PRO --- --- ASN TRP TYR ILE SER THR SER GUI ALA GLU ASN
bFGF aFGF IL-1
146 GLY PRO --- GLY GUN LYS ALA ILE LEU PHE LEU PRO MET SER ALA LYS E 124 TYR YS LEU GLY SER LYS 117 SER LYS LEU GLY PRO ARG HIS PHE --- GLY GLN LYS ALA ILE LEU PHE LEU PRO LEU PRO J LSERSERASP 140 P 246 MET PRO VAL PHE LEU GLY GLY THR LYS GLY GLY THR MET GLN PHE VAL SER SER 269 ASP ILE THR A
---
aao a a a a)
---
* * *a*a*a GI
FIG. 5. Alignment of the amino acid sequences of human bFGF (35) [sequence numbered to correspond to bovine bFGF (25)] and IL-113 (15, 26) suggested by the correspondence between their three-dimensional crystal structures. The sequence of bovine aFGF (15) is also included. The 50 residues whose a-carbons correspond with a rms discrepancy of 0.52 A are indicated with asterisks. Residues in bFGF that are indicated by the algorithm of Kabsch and Sander (36) as having a 13-sheet conformation are indicated (13). There are 10 such "3-sheet strands" shown in the figure. There are two additional segments, comprising residues 117-119 and 124-129 and indicated (J . . . 13), that do not meet the Kabsch and Sander criteria for 13-sheet strands, but, nevertheless, form the 10th and 11th of the 12 18-strands that comprise the overall framework of the bFGF and IL-1,8 structures.
correspond to Asn-27, Arg-120, and Lys-125 in the amino acid sequence of aFGF (Fig. 5) are, respectively, Asn-18, Lys-113, and Lys-118. The methylation of Lys-118 of aFGF (46), or its replacement by a glutamic acid (1), significantly reduces the affinity of aFGF for immobilized heparin. It seems likely that Asn-27, Arg-120, and Lys-125 on bFGF and Asn-18, Lys-113, and Lys-118 on aFGF constitute binding sites for heparin and other sulfated substrates. In contrast, however, the three corresponding amino acids in IL-1,3 (Fig. 5) are Gln-120, Gln-242, and Pro-247, making it very unlikely that this could be a sulfate or heparin binding site on the interleukin structure. Electron density, possibly compatible with a second bound sulfate ion, was observed in the bFGF crystal structure about 8 A from the first site. The apparent hydrogen-bonding groups include the main-chain amide of Leu-126 and the side chains of Lys-119 and Lys-129. In this case, however, the electron density is not as strong and as clearly defined as at the first site. Cysteine Locations There are four cysteines in bFGF, located at positions 25, 69, 87, and 92. Cys-25 and Cys-92 are highly conserved in different members of the FGF family (1, 4, 5). Whether this or another pair of cysteines might form a disulfide bond has been the subject of debate (1, 47-49). All four cysteines have been replaced with serines by two independent groups but with somewhat different results. Seno et al. (47) observed a decrease in biological activity, whereas Arakawa et al. (49) reported no significant effect on FGF activity. In the crystal structure of bFGF the electron density defining the positions
of Cys-25, -69, and -92 is clear. Both Cys-69 and Cys-92 are exposed to solvent, whereas Cys-25 is completely buried in a loop region. It seems clear that there is no possibility of disulfide formation between these three well-defined cysteines. Cys-25 is situated 21 A from both Cys-69 and Cys-92, and the distance between Cys-69 and Cys-92 is 19 A. The only cysteines situated close enough to possibly form a disulfide bond are Cys-87 and Cys-92, and the electron density in the vicinity of Cys-87 is extremely diffuse. These two residues, however, have never been suggested to form such a bond (47-49). Receptor Binding
The peptide fragments 24-68 and 106-115 of bFGF bind to heparin, interact with the bFGF receptor, and have agonist activity in mitogenic assays for bFGF (42). The first peptide has relatively low potency but the second displays all the features expected of a receptor-binding peptide. Fragment 106-115 begins in the middle of the 9th /-strand, makes a somewhat open loop on the surface of the bFGF molecule, and terminates at the beginning of the 10th /-strand (Figs. 3 and 5). Fragment 106-115 is also within the most extended segment of bFGF that structurally corresponds to IL-1p (Figs. 4a and 5). At face value this structural conservation within the putative receptor-binding region might suggest that the backbone conformation is common to a broad family of receptor-binding proteins and that the differences in amino acid sequence within this conserved motif could account for the discrimination between different receptors. It would be attractive to consider that receptor recognition by a conserved "strand-turn-strand" motif in bFGF, IL-101, and other
Biochemistry: Eriksson et A putative factors might be a counterpart to the use of the conserved "helix-turn-helix" motif in the recognition of specific binding sites on DNA (50). Further inspection, however, suggests that the basis of recognition and discrimination in the case of bFGF and IL-1,/ may be more complicated. Within the solvent-exposed loop region, residues 111-114 of bFGF form a type I turn (51). In contrast, the corresponding residues in IL-1,3, 233-236, form a type II turn. This is a significant difference in the geometry of the respective peptide backbones. Perhaps more dramatic is the apparent difference in this region between aFGF and bFGF. aFGF and bFGF bind to similar receptors, but with different affinity for different types of receptor species (52). According to the sequence alignment shown in Fig. 5, however, aFGF has two additional amino acids inserted at or near the position corresponding to the 112-113 peptide bond of bFGF. Therefore the loop in aFGF that corresponds to the 106-115 fragment of bFGF cannot have the same backbone conformation. It could be that the parts of aFGF and bFGF that contact receptor do not specifically include the region of the dipeptide insertion, but this remains to be determined. The presumed receptor-binding region of bFGF is about 25 A from the presumed heparin binding site and the two sites are on different faces of the molecule (Fig. 3). This suggests that receptor binding and the binding of heparin are likely to be more-or-less independent, as is also indicated by the finding that neutralizing antibodies inhibit the binding of bFGF to its receptor but not to heparin (53). The apparent separation of the heparin and receptor binding sites also suggests that the potentiating effect of heparin on the mitogenic activity of aFGF and its inhibition of the growthpromoting activity of bFGF (42-44) are due to indirect effects. Bulky polyanionic compounds, such as suramin, that inhibit the receptor binding of both aFGF and bFGF (54) could do so by binding to FGF and preventing access to the receptor-binding region or by causing a conformational change in the FGF molecule. We are most grateful to Dr. Dale Tronrud, Dr. Jim Remington, and especially Dr. Steve Roderick for extensive help and advice with the structure determination. A.E.E. extends special thanks to Dr. Sherry Mowbray, Uppsala University, Sweden, for her suggestion of using macroseeding as a method for obtaining large beautiful crystals. We thank Drs. J. Priestle and M. Grutter, CIBA-Geigy, for supplying the refined coordinates of IL-1,t (also available from the Brookhaven Protein Data Bank). A.E.E. acknowledges the support of the Swed-
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