Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members

Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/betaKlotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between beta strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the beta1-beta2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/betaKlotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors.     

Mutations in the heparin-binding domains of human basic fibroblast growth factor alter its biological activity

Eleven structural analogues of human basic fibroblast growth factor (bFGF) have been prepared by site-directed mutagenesis of a synthetic bFGF gene to examine the effect of amino acid substitutions in the three putative heparin-binding domains on FGF's biological activity. After expression in Escherichia coli, the mutant proteins were purified to homogeneity by use of heparin-Sepharose chromatography and analyzed for their ability to stimulate DNA synthesis in human foreskin fibroblasts. Recombinant human bFGF 1-146 and [Ala69,Ser87]bFGF, an analogue where two of the four cysteines had been replaced by alanine and serine, were equipotent to standard bovine basic fibroblast growth factor. Substitution of aspartic acid-19 by arginine in the first heparin-binding domain yielded a molecule that stimulated a higher total mitogenic response in fibroblasts as compared to bFGF. In addition, replacement of either arginine-107 in the second domain or glutamine-123 in the third domain with glutamic acid resulted in compounds that were 2 and 4 times more potent than bFGF. In contrast, substitution of arginine-107 with isoleucine reduced the activity of the molecule by 100-fold. Combination of domain substitutions to generate the [Glu107,123]bFGF and [Arg19,Lys123,126]bFGF mutants did not show any additivity of the mutations on biological activity. Alterations in the biological activity of the analogues was dependent on both the site of and the type of modification. Increased positive charge in the first domain and increased negative charge in the second and third domains enhanced biological potency. The altered activities of the derivatives appear to be due in part to changes in the affinity of the analogues for heparin. We conclude that changes in all three of the putative heparin-binding domains result in altered mitogenic activity and heparin interaction of basic fibroblast growth factor.     

In-gel ligand blotting with 125I-heparin for detection of heparin-binding growth factors

125I-labeled heparin was used to detect basic fibroblast growth factor (bFGF) in crude tumor cell extracts after separation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. 125I-labeled heparin bound avidly to native recombinant bFGF immobilized on nitrocellulose and eluted with 1.5-2.0 M NaCl. However, Western transfer of bFGF to nitrocellulose after SDS-polyacrylamide gel electrophoresis destroyed heparin-binding ability. In contrast, bFGF was detected by incubation of the polyacrylamide gels directly with 125I-labeled heparin in a gel overly technique. Heparin affinity and NaCl elution pattern from bFGF in gel were similar to those observed for native bFGF spotted on nitrocellulose. This procedure permitted detection of bFGF in crude extracts of a human astrocytoma cell line. In view of the rapid growth of the heparin-binding fibroblast growth factor gene family, this technique should prove useful for the rapid and sensitive detection of other heparin-binding growth factors.     

Binding of heparin to basic fibroblast growth factor induces a conformational change.

The binding of heparin to basic fibroblast growth factor (bFGF) induces a small but highly reproducible conformational change observable in the amide I region of the protein's infrared spectrum. The observed spectral changes suggest that the conformational change is highly localized most likely in the beta-turn regions of the bFGF molecule. Heparan sulfate, a component of the endothelial extracellular matrix, was also observed to bind to bFGF and induce a similar conformational change to that observed for heparin. Further, sucrose octasulfate, a compound which mimics the effects of heparin biologically, was also observed to induce this same conformational change. This spectroscopically observable change has allowed us to probe the functional determinants necessary for heparin to bind the bFGF and to induce the observed conformational change. We have determined the effects of binding of various monomeric and polymeric, sulfated and nonsulfated glycosaminoglycans and carbohydrate compounds. The results indicate that the binding of heparin involves highly specific interactions. Further, heparin was observed to greatly increase the thermal stability of bFGF, raising the Tm by 25 degrees C. Sucrose octasulfate was also able to enhance the thermal stability of bFGF, but not to the same extent as heparin.     

Origin of the anomalous circular dichroism spectra of many apomyoglobin mutants

Several authors have reported that many sperm whale apomyoglobin mutants show anomalous circular dichroism spectra. These mutants have a low molar ellipticity compared to the wild-type protein but in several cases have the same stability of unfolding. A model in which native apomyoglobin is not folded in the same manner as that in other proteins and in which mutants show progressive reductions in their degree of folding has been suggested to explain this phenomenon. However, nuclear magnetic resonance of the native apomyoglobin conformation has shown that this state is folded and compact, raising the possibility that the anomalous circular dichroism spectra could have another explanation. We studied several mutants with anomalous circular dichroism spectra and found that these proteins were all contaminated with nucleic acid that contributed to the ultraviolet absorption and caused uncertainty in the determination of protein concentration. The resulting overestimation of the concentration of apomyoglobin explains the phenomenon of anomalous circular dichroism spectra. We describe a procedure to remove the contaminant nucleic acid which yields accurate protein concentration measurements and provides the normal circular dichroism spectra. Our findings support a well-structured native conformation for apomyoglobin and may also be of the interest to scientists working with the purification of recombinant proteins.     

Interaction of heparin with human basic fibroblast growth factor: protection of the angiogenic protein from proteolytic degradation by a glycosaminoglycan

Fibroblast growth factors (FGF) are a family of heparin-binding angiogenic polypeptide mitogens. In the presence of heparin, recombinant human basic fibroblast growth factor (bFGF) is fully protected from tryptic digestion and partially protected from chymotryptic digestion. Complete protection of bFGF by heparin is achieved at ratios of growth factor:heparin of approximately 10 or less (w/w). The protection requires bioactive bFGF because inactivated bFGF is rapidly degraded by trypsin or chymotrypsin in the presence of heparin. The bFGF-heparin interaction forms hydrophobic complexes which become insoluble in aqueous buffers at bFGF:heparin ratios of 8 to 10 (w/w). The heparin was found to bind up to a tenfold excess of bFGF on a weight basis. bFGF in the presence of heparin is as active as bFGF alone in inducing 3H-thymidine incorporation into Swiss 3T3 fibroblast DNA.     

Molecular cloning, overexpression, and characterization of the ligand-binding D2 domain of fibroblast growth factor receptor

Fibroblast growth factors (FGFs) regulate a wide range of important cellular processes. The biological activities of FGFs are mediated by cell surface receptors (FGFRs). In the present study for the first time we report the cloning, expression, and characterization of the ligand (FGF)-binding D2 domain of human FGFR2. D2 domain is expressed in Escherichia coli in high yields (10 mg/L) as inclusion bodies. The protein is recovered by dissolving the inclusion bodies in 8 M urea and subsequently refolding on nickel affinity column. The protein is purified (to approximately 97% purity) to homogeneity using heparin-Sepharose affinity column. Far-UV circular dichroism data and chemical shift index plot based on 1H-alpha, 13C-alpha, 13C-beta, and 13carbonyl group chemical shifts suggest that D2 domain is an all beta-sheet protein consisting of 9 beta-strands. Isothermal titration calorimetry and equilibrium urea unfolding experiments show that recombinant D2 domain is in a biologically active conformation and binds strongly to its ligand (FGF) and to the heparin analog, sucrose octasulfate (SOS). Using a variety of triple resonance NMR experiments, complete assignment of 1H, 15N, and 13C resonances in D2 domain has been accomplished. The findings of the present study not only pave way for an in-depth investigation of the molecular mechanism(s) underlying the activation of FGF signaling but also provide avenues for the rational design of potent inhibitors against FGF-mediated pathogenesis.     

Densimetric determination of equilibrium binding of sucrose octasulfate with basic fibroblast growth factor

Fibroblast growth factors (FGFs) strongly bind to heparin and are thereby stabilized against deactivation and proteolytic cleavage. Sucrose octasulfate (SOS), which has a chemical structure resembling the repeating unit of heparin, has also been shown to enhance stability of basic FGF against thermal denaturation and to induce a small conformational change. We have examined SOS binding to bFGF using equilibrium dialysis. The difference in SOS concentration across the dialysis membrane was measured using a precision density meter, since the density of SOS differs greatly from that of water. With care, this densimetric technique can measure binding with a precision of ± 0.1 mol/mol using about 2 mg/ml of protein. These results show that the binding saturates at 2 mol of SOS per mole of bFGF as the SOS concentration increases to 3.6 mM or higher. The effect of SOS on the thermal stability of bFGF was examined using denaturation at a constant heating rate, by both turbidity and differential scanning calorimetry. Since the thermal denaturation is irreversible, the temperature where aggregation abruptly increases was taken to indicate the onset of denaturation. This temperature increased by 12°C as the SOS concentration increased from 0.018 to 3.6 mM and remained constant above 3.6 mM, consistent with our binding data if the binding is specific to the native state.     

Ligand specificity and heparin dependence of fibroblast growth factor receptors 1 and 3.

The heparin-binding growth factors include a family of seven structurally related proteins that can potentially interact with four known high affinity receptors. We have cloned the murine homologues of fibroblast growth factor receptors 1 and 3 (mFR1 and mFR3). To define the ligand specificity of these receptors, we have characterized their binding properties with respect to acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) and their biologic activity with respect to aFGF, bFGF, FGF-4/K-FGF, and FGF-5. Unlike mFR1, which binds both aFGF and bFGF, mFR3 preferentially binds aFGF. mFR3-mediated mitogenicity also favors aFGF and FGF-4 with a 10-12-fold lower response to bFGF and no response to FGF-5. Both receptor binding and growth factor-mediated mitogenicity are dependent on heparin. Heparin-binding growth factor activity can thus be regulated by proteoglycans and by the type of FGF receptor expressed on the target cell.     

Interactions of multiple heparin binding growth factors with neuropilin-1 and potentiation of the activity of fibroblast growth factor-2

The hypothesis that neuropilin-1 (Npn-1) may interact with heparin-binding proteins other than vascular endothelial growth factor has been tested using an optical biosensor-based binding assay. The results show that fibroblast growth factor (FGF) 1, 2, 4, and 7, FGF receptor 1, hepatocyte growth factor/scatter factor (HGF/SF), FGF-binding protein, normal protease sensitive form of prion protein, antithrombin III, and Npn-1 itself are all able to interact with Npn-1 immobilized on the sensor surface. FGF-2, FGF-4, and HGF/SF are also shown to interact with Npn-1 in a solution assay. Moreover, these protein-protein interactions are dependent on the ionic strength of the medium and are inhibited by heparin, and the kinetics of binding of FGF-2, FGF-4 and HGF/SF to Npn-1 are characterized by fast association rate constants (270,000-1,600,000 m(-1) s(-1)). These results suggest that Npn-1 possesses a "heparin" mimetic site that is able to interact at least in part through ionic bonding with the heparin binding site on many of the proteins studied. Npn-1 was also found to potentiate the growth stimulatory activity of FGF-2 on human umbilical vein endothelial cells, indicating that Npn-1 may not just bind but also regulate the activity of heparin-binding proteins.     

NMR characterization of the electrostatic interaction of the basic residues in HDGF and FGF2 during heparin binding

Electrostatic interaction is a major driving force in the binding of proteins to highly acidic glycosaminoglycan, such as heparin. Although NMR backbone chemical shifts have generally been used to identify the heparin-binding site on a protein, however, there is no correlation between the binding free energies and the perturbed backbone chemical shifts for individual residues. The binding event occurs at the end of a side chain of basic residue, and does not require causing significant alterations in the backbone environment at a distance of multiple bonds. We used the H₂CN NMR pulse sequence to detect heparin binding through the side-chain resonances Hε–Cε–Nζ of Lys and Hδ–Cδ–Nε of Arg in the two proteins of hepatoma-derived growth factor (HDGF) and basic fibroblast growth factor (FGF2). H₂CN titration experiments revealed chemical shift perturbations in the side chains, which were correlated with the free energy changes in various mutants. The residues K19 in HDGF and K125 in FGF2 demonstrated the most significant perturbations, consistent with our previous observation that the two residues are crucial for binding. The result suggests that H₂CN NMR provides a precise evaluation for the electrostatic interactions. The discrepancy observed between backbone and side chain chemical shifts is correlated to the solvent accessibility of residues that the K19 and K125 backbones are highly buried with the restricted backbone conformation and are not strongly affected by the events at the end of the side chains.     

Xylosyltransferase I acceptor properties of fibroblast growth factor and its fragment bFGF (1-24)

Human basic fibroblast growth factor (bFGF) is a heparin-binding growth factor containing a G-S-G-motif which is a potential recognition sequence of xylosyltransferase I (XT-I). Here, we show that the recombinant human bFGF was xylosylated in vitro by human XT-I and that the fragment bFGF (1-24) is a good XT-I acceptor (K(m) = 20.8 microM for native XT-I and K(m) = 22.3 microM for recombinant XT-I). MALDI and MALDI-PSD time-of-flight mass spectrometric analyses of the xylosylated bFGF protein demonstrate the transfer of xylose to the serine residue of the G-S-G-motif in the amino terminal end of bFGF. The peptide bFGF (1-24) is well suitable as an acceptor substrate for XT-I and can be used in a radiochemical assay to measure the XT-I activity in cell culture supernatant and human body fluids, respectively. Furthermore, we could demonstrate that the XT-I interacts strongly with heparin and that this glycosaminoglycan is a predominantly non-competitive inhibitor of the enzyme using the fragment bFGF (1-24) as xylose acceptor.     

Identification and dynamics of a heparin-binding site in hepatocyte growth factor.

Hepatocyte growth factor (HGF) is a heparin-binding, multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. Heparin or closely related heparan sulfate has profound effects on HGF signaling. A heparin-binding site in the N-terminal (N) domain of HGF was proposed on the basis of the clustering of surface positive charges [Zhou, H., Mazzulla, M. J., Kaufman, J. D., Stahl, S. J., Wingfield, P. T., Rubin, J. S., Bottaro, D. P., and Byrd, R. A. (1998) Structure 6, 109-116]. In the present study, we confirmed this binding site in a heparin titration experiment monitored by nuclear magnetic resonance spectroscopy, and we estimated the apparent dissociation constant (K(d)) of the heparin-protein complex by NMR and fluorescence techniques. The primary heparin-binding site is composed of Lys60, Lys62, and Arg73, with additional contributions from the adjacent Arg76, Lys78, and N-terminal basic residues. The K(d) of binding is in the micromolar range. A heparin disaccharide analogue, sucrose octasulfate, binds with similar affinity to the N domain and to a naturally occurring HGF isoform, NK1, at nearly the same region as in heparin binding. (15)N relaxation data indicate structural flexibility on a microsecond-to-millisecond time scale around the primary binding site in the N domain. This flexibility appears to be dramatically reduced by ligand binding. On the basis of the NK1 crystal structure, we propose a model in which heparin binds to the two primary binding sites and the N-terminal regions of the N domains and stabilizes an NK1 dimer.     

Nature of Interaction between basic fibroblast growth factor and the antiangiogenic drug 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino])-bis-(1,3-naphtalene disulfonate). II. Removal of polar interactions affects protein folding

Fibroblast growth factor-2 (basic FGF), a potent inducer of angiogenesis, and the naphthalene sulfonic distamycin A derivative, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino])-bis-(1,3-naphtalene disulfonate) (PNU145156E), which exhibits in vivo antiangiogenic activity, form a tight reversible (1:1) complex. PNU145156E binds to the heparin and the selenate-binding sites on bFGF. The cis bFGF-heparin (2:1) complex, essential for the activation of the angiogenic process, is thus prevented. The nature of the forces involved in bFGF:PNU145156E complex, using the wild-type and the K128Q, K138Q, K134Q, and K128Q-K138Q point mutated bFGFs was sought. Based on thermodynamic analysis of the complexation constants, protein temperature stability profiles by ultraviolet absorption, circular dichroism measurements, fluorescence F?rster energy-transfer, and anisotropy studies, in harmony with the published x-ray crystallographic structure, the following molecular interactions are proposed: reduced coulombic interactions, hence loosening of the complex by the removal of charged polar groups from the bFGF-heparin binding cleft resulted in decreased binding constants and in a change in the binding mode from polar to nonpolar. Concomitantly, upon mutation, the protein was rendered more compact, less flexible, and less aqueously exposed compared with the wild type. These were further pronounced with the double mutant: weaker dominantly nonpolar protein-drug interactions were accompanied by conspicuous folding. With heparin, however, wild-type bFGF forms a tighter complex with a more compact structure.     

Structural basis for activation of fibroblast growth factor signaling by sucrose octasulfate

Sucrose octasulfate (SOS) is believed to stimulate fibroblast growth factor (FGF) signaling by binding and stabilizing FGFs. In this report, we show that SOS induces FGF-dependent dimerization of FGF receptors (FGFRs). The crystal structure of the dimeric FGF2-FGFR1-SOS complex at 2.6-A resolution reveals a symmetric assemblage of two 1:1:1 FGF2-FGFR1-SOS ternary complexes. Within each ternary complex SOS binds to FGF and FGFR and thereby increases FGF-FGFR affinity. SOS also interacts with the adjoining FGFR and thereby promotes protein-protein interactions that stabilize dimerization. This structural finding is supported by the inability of selectively desulfated SOS molecules to promote receptor dimerization. Thus, we propose that SOS potentiates FGF signaling by imitating the dual role of heparin in increasing FGF-FGFR affinity and promoting receptor dimerization. Hence, the dimeric FGF-FGFR-SOS structure substantiates the recently proposed "two-end" model, by which heparin induces FGF-FGFR dimerization. Moreover, the FGF-FGFR-SOS structure provides an attractive template for the development of easily synthesized SOS-related heparin agonists and antagonists that may hold therapeutic potential.     

Basic fibroblast growth factor is a beta-rich protein.

The conformation of the 153-residue form of human basic fibroblast growth factor (bFGF) was studied with circular dichroism (CD) and sequence prediction methods. The far-UV CD spectrum with a minimum at 202 nm resembled that of an unordered polypeptide/protein or a protein rich in distorted antiparallel -sheets. Analysis of the CD spectrum by the least-squares method of Changet al. (1978) and the CONTIN program of Provencher and Glöckner (1981) suggested that about one half of the molecule consisted of -sheet and there was no -helix. These estimates agreed with the prediction by the sequence method of Garnieret al. (1978) using decision constants based on CD results. bFGF had an unusual CD band at 187 nm, which disappeared upon ionization of Tyr side chains atpH 11.7. It also had another unusual property of irreversibly converting the CD spectrum to a helix-like one with a double minimum at 205 and 215 and a maximum at 189 nm upon heating the solution to above 55°C. The helicity was also enhanced in trifluoroethanol and in sodium dodecyl sulfate. The mutant bFGF in which cysteines 76 and 94 were replaced by serine residues had essentially the same properties as the wild-type.     

Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor.

The role of low affinity, heparin-like binding sites for basic fibroblast growth factor (bFGF) was investigated in CHO cells mutant in their metabolism of glycosaminoglycans. Heparan sulfate-deficient mutants transfected to express a cloned mouse FGF receptor cDNA are not able to bind bFGF. It is demonstrated that free heparin and heparan sulfate can reconstitute a low affinity receptor that is, in turn, required for the high affinity binding of bFGF. These studies suggest that the low affinity receptor is an accessory molecule required for binding of bFGF to the high affinity site. Such an obligatory interaction of low and high affinity FGF receptors suggests a physiological role for heparin-like, low affinity receptors and constitutes a novel mechanism for the regulation of growth factor-receptor interactions.     

Effect of different immunosuppressive drugs on calcineurin and its mutants

Several mutants in Loop7 region and near Loop7 region of calcineurin A (CN A) subunit have been constructed and purified using site-directed mutagenesis. Their phosphatase activity and the corresponding solution conformation were examined. Their phosphatase activities between wild-type CN and mutants were compared to identify the interaction of different immuno-suppressive drugs with CN. The results showed that the phosphatase activities of the mutants at Loop7 were much higher than the one of wild-type CN. Furthermore, circular dichroism spectra of the mutants revealed that their solution conformations gave rise in changes in native structure of the protein. Cyclophilin-CyclosporinA (CyP-CsA) significantly inhibited the phosphatase activity of wild-type CN, and had no effects on the phosphatase activity of mutants in Loop7 region, which indicates that the site-directed mutagenesis at Loop7 region made a significant change in the interaction between CyP-CsA and CN. Examination of the activities of these     

Secondary structure of the entomocidal toxin from Bacillus thuringiensis subsp. kurstaki HD-73

The secondary structure of the toxin fromBacillus thuringiensis subsp.kurstaki (Btk) HD-73 was estimated by Raman, infrared, and circular dichroism spectroscopy, and by predictive methods. Circular dichroism and infrared spectroscopy gave an estimate of 33–40% -helix, whereas Raman and predictive methods gave approximately 20%. Raman and circular dichroism spectra, as well as predictive methods, indicated that the toxin contains 32–40% -sheet structure, whereas infrared spectroscopy gave a slightly lower estimate. Thus, all of these approaches are in agreement that the native conformation of Btk HD-73 toxin is highly folded and contains considerable amounts of both -helical and -sheet structures. No significant differences were detected in the secondary structure of the toxin either in solution or as a hydrated pellet.     

Vibrational circular dichroism studies of epidermal growth factor and basic fibroblast growth factor.

Vibrational circular dichroism (VCD) studies are reported for two unrelated recombinant growth factor proteins: epidermal growth factor and basic fibroblast growth factor (bFGF). NMR, electronic CD, and bFGF X-ray studies indicate that these two proteins are primarily composed of beta-sheet and loop secondary structure elements with no detectable alpha-helices. Two reports on solution conformation of these proteins using FTIR absorption spectroscopy with subsequent resolution enhancement confirmed the presence of a large fraction of a beta-sheet conformation but in addition indicated the presence of large absorption bands in the 1650-1656 cm-1 region, which are typically assigned to alpha-helices. The VCD spectra of both proteins have band shapes that strongly resemble those of other high beta-sheet fraction proteins, such as the trypsin family of proteins. Quantitative analysis of the VCD spectra also indicates that these proteins are predominantly in beta-sheet and extended ("other") conformations with very little alpha-helix fraction. These results agree with the CD interpretation and affirm that the FTIR peaks in the region 1650-1656 cm-1 can be assigned to loops. This study provides an example of the limitations of using FTIR frequencies alone for examination of protein secondary structure.