Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1993 Aug;2(8):1274–1284. doi: 10.1002/pro.5560020810

Refinement of the structure of human basic fibroblast growth factor at 1.6 A resolution and analysis of presumed heparin binding sites by selenate substitution.

A E Eriksson 1, L S Cousens 1, B W Matthews 1
PMCID: PMC2142437  PMID: 7691311

Abstract

The three-dimensional structure of human basic fibroblast growth factor has been refined to a crystallographic residual of 16.1% at 1.6 A resolution. The structure has a Kunitz-type fold and is composed of 12 antiparallel beta-strands, 6 of which form a beta-barrel. One bound sulfate ion has been identified in the model, hydrogen bonded to the side chains of Asn 27, Arg 120, and Lys 125. The side chain of Arg 120 has two conformations, both of which permit hydrogen bonds to the sulfate. This sulfate binding site has been suggested as the binding site for heparin (Eriksson, A.E., Cousens, L.S., Weaver, L.H., & Matthews, B.W., 1991, Proc. Natl. Acad. Sci. USA 88, 3441-3445). Two beta-mercaptoethanol (BME) molecules are also included in the model, each forming a disulfide bond to the S gamma atoms of Cys 69 and Cys 92, respectively. The side chain of Cys 92 has two conformations of which only one can bind BME. Therefore the BME molecule is half occupied at this site. The locations of possible sulfate binding sites on the protein were examined by replacing the ammonium sulfate in the crystallization medium with ammonium selenate. Diffraction data were measured to 2.2 A resolution and the structure refined to an R-factor of 13.8%. The binding of the more electron-dense selenate ion was identified at two positions. One position was identical to the sulfate binding site identified previously. The second selenate binding site, which is of lower occupancy, is situated 5.6 A from the first. This ion is hydrogen bonded by the side chain of Lys 135 and Arg 120. Thus the side chain of Arg 120 binds two selenate ions simultaneously. It is suggested that the observed second selenate binding site should also be considered as a possible binding site for heparin, or that both selenate binding sites might simultaneously contribute to the binding of heparin.

Full Text

The Full Text of this article is available as a PDF (2.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abraham J. A., Whang J. L., Tumolo A., Mergia A., Friedman J., Gospodarowicz D., Fiddes J. C. Human basic fibroblast growth factor: nucleotide sequence and genomic organization. EMBO J. 1986 Oct;5(10):2523–2528. doi: 10.1002/j.1460-2075.1986.tb04530.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ago H., Kitagawa Y., Fujishima A., Matsuura Y., Katsube Y. Crystal structure of basic fibroblast growth factor at 1.6 A resolution. J Biochem. 1991 Sep;110(3):360–363. doi: 10.1093/oxfordjournals.jbchem.a123586. [DOI] [PubMed] [Google Scholar]
  3. Baird A., Klagsbrun M. The fibroblast growth factor family. Cancer Cells. 1991 Jun;3(6):239–243. [PubMed] [Google Scholar]
  4. Baird A., Schubert D., Ling N., Guillemin R. Receptor- and heparin-binding domains of basic fibroblast growth factor. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2324–2328. doi: 10.1073/pnas.85.7.2324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burgess W. H., Dionne C. A., Kaplow J., Mudd R., Friesel R., Zilberstein A., Schlessinger J., Jaye M. Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase. Mol Cell Biol. 1990 Sep;10(9):4770–4777. doi: 10.1128/mcb.10.9.4770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burgess W. H., Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem. 1989;58:575–606. doi: 10.1146/annurev.bi.58.070189.003043. [DOI] [PubMed] [Google Scholar]
  7. Burgess W. H., Shaheen A. M., Ravera M., Jaye M., Donohue P. J., Winkles J. A. Possible dissociation of the heparin-binding and mitogenic activities of heparin-binding (acidic fibroblast) growth factor-1 from its receptor-binding activities by site-directed mutagenesis of a single lysine residue. J Cell Biol. 1990 Nov;111(5 Pt 1):2129–2138. doi: 10.1083/jcb.111.5.2129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Caccia P., Nitti G., Cletini O., Pucci P., Ruoppolo M., Bertolero F., Valsasina B., Roletto F., Cristiani C., Cauet G. Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues. Eur J Biochem. 1992 Mar 1;204(2):649–655. doi: 10.1111/j.1432-1033.1992.tb16678.x. [DOI] [PubMed] [Google Scholar]
  9. Coughlin S. R., Barr P. J., Cousens L. S., Fretto L. J., Williams L. T. Acidic and basic fibroblast growth factors stimulate tyrosine kinase activity in vivo. J Biol Chem. 1988 Jan 15;263(2):988–993. [PubMed] [Google Scholar]
  10. Damon D. H., Lobb R. R., D'Amore P. A., Wagner J. A. Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half-life. J Cell Physiol. 1989 Feb;138(2):221–226. doi: 10.1002/jcp.1041380202. [DOI] [PubMed] [Google Scholar]
  11. Delli Bovi P., Curatola A. M., Kern F. G., Greco A., Ittmann M., Basilico C. An oncogene isolated by transfection of Kaposi's sarcoma DNA encodes a growth factor that is a member of the FGF family. Cell. 1987 Aug 28;50(5):729–737. doi: 10.1016/0092-8674(87)90331-x. [DOI] [PubMed] [Google Scholar]
  12. Dickson C., Peters G. Potential oncogene product related to growth factors. 1987 Apr 30-May 6Nature. 326(6116):833–833. doi: 10.1038/326833a0. [DOI] [PubMed] [Google Scholar]
  13. Eriksson A. E., Cousens L. S., Weaver L. H., Matthews B. W. Three-dimensional structure of human basic fibroblast growth factor. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3441–3445. doi: 10.1073/pnas.88.8.3441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Esch F., Baird A., Ling N., Ueno N., Hill F., Denoroy L., Klepper R., Gospodarowicz D., Böhlen P., Guillemin R. Primary structure of bovine pituitary basic fibroblast growth factor (FGF) and comparison with the amino-terminal sequence of bovine brain acidic FGF. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6507–6511. doi: 10.1073/pnas.82.19.6507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Finch P. W., Rubin J. S., Miki T., Ron D., Aaronson S. A. Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science. 1989 Aug 18;245(4919):752–755. doi: 10.1126/science.2475908. [DOI] [PubMed] [Google Scholar]
  16. Finzel B. C., Clancy L. L., Holland D. R., Muchmore S. W., Watenpaugh K. D., Einspahr H. M. Crystal structure of recombinant human interleukin-1 beta at 2.0 A resolution. J Mol Biol. 1989 Oct 20;209(4):779–791. doi: 10.1016/0022-2836(89)90606-2. [DOI] [PubMed] [Google Scholar]
  17. Friesel R., Burgess W. H., Maciag T. Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells. Mol Cell Biol. 1989 May;9(5):1857–1865. doi: 10.1128/mcb.9.5.1857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gimenez-Gallego G., Rodkey J., Bennett C., Rios-Candelore M., DiSalvo J., Thomas K. Brain-derived acidic fibroblast growth factor: complete amino acid sequence and homologies. Science. 1985 Dec 20;230(4732):1385–1388. doi: 10.1126/science.4071057. [DOI] [PubMed] [Google Scholar]
  19. Gospodarowicz D., Cheng J. Heparin protects basic and acidic FGF from inactivation. J Cell Physiol. 1986 Sep;128(3):475–484. doi: 10.1002/jcp.1041280317. [DOI] [PubMed] [Google Scholar]
  20. Graves B. J., Hatada M. H., Hendrickson W. A., Miller J. K., Madison V. S., Satow Y. Structure of interleukin 1 alpha at 2.7-A resolution. Biochemistry. 1990 Mar 20;29(11):2679–2684. doi: 10.1021/bi00463a009. [DOI] [PubMed] [Google Scholar]
  21. Habazettl J., Gondol D., Wiltscheck R., Otlewski J., Schleicher M., Holak T. A. Structure of hisactophilin is similar to interleukin-1 beta and fibroblast growth factor. Nature. 1992 Oct 29;359(6398):855–858. doi: 10.1038/359855a0. [DOI] [PubMed] [Google Scholar]
  22. Harper J. W., Lobb R. R. Reductive methylation of lysine residues in acidic fibroblast growth factor: effect on mitogenic activity and heparin affinity. Biochemistry. 1988 Jan 26;27(2):671–678. doi: 10.1021/bi00402a027. [DOI] [PubMed] [Google Scholar]
  23. Huang S. S., Huang J. S. Association of bovine brain-derived growth factor receptor with protein tyrosine kinase activity. J Biol Chem. 1986 Jul 25;261(21):9568–9571. [PubMed] [Google Scholar]
  24. Janin J., Wodak S. Conformation of amino acid side-chains in proteins. J Mol Biol. 1978 Nov 5;125(3):357–386. doi: 10.1016/0022-2836(78)90408-4. [DOI] [PubMed] [Google Scholar]
  25. Jaye M., Howk R., Burgess W., Ricca G. A., Chiu I. M., Ravera M. W., O'Brien S. J., Modi W. S., Maciag T., Drohan W. N. Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization. Science. 1986 Aug 1;233(4763):541–545. doi: 10.1126/science.3523756. [DOI] [PubMed] [Google Scholar]
  26. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  27. Kan M., Wang F., Xu J., Crabb J. W., Hou J., McKeehan W. L. An essential heparin-binding domain in the fibroblast growth factor receptor kinase. Science. 1993 Mar 26;259(5103):1918–1921. doi: 10.1126/science.8456318. [DOI] [PubMed] [Google Scholar]
  28. Maciag T., Mehlman T., Friesel R., Schreiber A. B. Heparin binds endothelial cell growth factor, the principal endothelial cell mitogen in bovine brain. Science. 1984 Aug 31;225(4665):932–935. doi: 10.1126/science.6382607. [DOI] [PubMed] [Google Scholar]
  29. Marics I., Adelaide J., Raybaud F., Mattei M. G., Coulier F., Planche J., de Lapeyriere O., Birnbaum D. Characterization of the HST-related FGF.6 gene, a new member of the fibroblast growth factor gene family. Oncogene. 1989 Mar;4(3):335–340. [PubMed] [Google Scholar]
  30. McLachlan A. D. Three-fold structural pattern in the soybean trypsin inhibitor (Kunitz). J Mol Biol. 1979 Oct 9;133(4):557–563. doi: 10.1016/0022-2836(79)90408-x. [DOI] [PubMed] [Google Scholar]
  31. Murzin A. G., Lesk A. M., Chothia C. beta-Trefoil fold. Patterns of structure and sequence in the Kunitz inhibitors interleukins-1 beta and 1 alpha and fibroblast growth factors. J Mol Biol. 1992 Jan 20;223(2):531–543. doi: 10.1016/0022-2836(92)90668-a. [DOI] [PubMed] [Google Scholar]
  32. Musci T. J., Amaya E., Kirschner M. W. Regulation of the fibroblast growth factor receptor in early Xenopus embryos. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8365–8369. doi: 10.1073/pnas.87.21.8365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Neufeld G., Gospodarowicz D. Basic and acidic fibroblast growth factors interact with the same cell surface receptors. J Biol Chem. 1986 Apr 25;261(12):5631–5637. [PubMed] [Google Scholar]
  34. Neufeld G., Gospodarowicz D., Dodge L., Fujii D. K. Heparin modulation of the neurotropic effects of acidic and basic fibroblast growth factors and nerve growth factor on PC12 cells. J Cell Physiol. 1987 Apr;131(1):131–140. doi: 10.1002/jcp.1041310119. [DOI] [PubMed] [Google Scholar]
  35. Nurcombe V., Ford M. D., Wildschut J. A., Bartlett P. F. Developmental regulation of neural response to FGF-1 and FGF-2 by heparan sulfate proteoglycan. Science. 1993 Apr 2;260(5104):103–106. doi: 10.1126/science.7682010. [DOI] [PubMed] [Google Scholar]
  36. Priestle J. P., Schär H. P., Grütter M. G. Crystal structure of the cytokine interleukin-1 beta. EMBO J. 1988 Feb;7(2):339–343. doi: 10.1002/j.1460-2075.1988.tb02818.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Saksela O., Moscatelli D., Sommer A., Rifkin D. B. Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation. J Cell Biol. 1988 Aug;107(2):743–751. doi: 10.1083/jcb.107.2.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schreiber A. B., Kenney J., Kowalski W. J., Friesel R., Mehlman T., Maciag T. Interaction of endothelial cell growth factor with heparin: characterization by receptor and antibody recognition. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6138–6142. doi: 10.1073/pnas.82.18.6138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shing Y., Folkman J., Sullivan R., Butterfield C., Murray J., Klagsbrun M. Heparin affinity: purification of a tumor-derived capillary endothelial cell growth factor. Science. 1984 Mar 23;223(4642):1296–1299. doi: 10.1126/science.6199844. [DOI] [PubMed] [Google Scholar]
  40. Sweet R. M., Wright H. T., Janin J., Chothia C. H., Blow D. M. Crystal structure of the complex of porcine trypsin with soybean trypsin inhibitor (Kunitz) at 2.6-A resolution. Biochemistry. 1974 Sep 24;13(20):4212–4228. doi: 10.1021/bi00717a024. [DOI] [PubMed] [Google Scholar]
  41. Terranova V. P., DiFlorio R., Lyall R. M., Hic S., Friesel R., Maciag T. Human endothelial cells are chemotactic to endothelial cell growth factor and heparin. J Cell Biol. 1985 Dec;101(6):2330–2334. doi: 10.1083/jcb.101.6.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Thompson S. A. The disulfide structure of bovine pituitary basic fibroblast growth factor. J Biol Chem. 1992 Feb 5;267(4):2269–2273. [PubMed] [Google Scholar]
  43. Thornton S. C., Mueller S. N., Levine E. M. Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science. 1983 Nov 11;222(4624):623–625. doi: 10.1126/science.6635659. [DOI] [PubMed] [Google Scholar]
  44. Tronrud D. E. Conjugate-direction minimization: an improved method for the refinement of macromolecules. Acta Crystallogr A. 1992 Nov 1;48(Pt 6):912–916. doi: 10.1107/s0108767392005415. [DOI] [PubMed] [Google Scholar]
  45. Uhlrich S., Lagente O., Lenfant M., Courtois Y. Effect of heparin on the stimulation of non-vascular cells by human acidic and basic FGF. Biochem Biophys Res Commun. 1986 Jun 30;137(3):1205–1213. doi: 10.1016/0006-291x(86)90353-0. [DOI] [PubMed] [Google Scholar]
  46. Vainikka S., Partanen J., Bellosta P., Coulier F., Birnbaum D., Basilico C., Jaye M., Alitalo K. Fibroblast growth factor receptor-4 shows novel features in genomic structure, ligand binding and signal transduction. EMBO J. 1992 Dec;11(12):4273–4280. doi: 10.1002/j.1460-2075.1992.tb05526.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Zemke K. J., Müller-Fahrnow A., Jany K. D., Pal G. P., Saenger W. The three-dimensional structure of the bifunctional proteinase K/alpha-amylase inhibitor from wheat (PK13) at 2.5 A resolution. FEBS Lett. 1991 Feb 25;279(2):240–242. doi: 10.1016/0014-5793(91)80158-y. [DOI] [PubMed] [Google Scholar]
  48. Zhan X., Bates B., Hu X. G., Goldfarb M. The human FGF-5 oncogene encodes a novel protein related to fibroblast growth factors. Mol Cell Biol. 1988 Aug;8(8):3487–3495. doi: 10.1128/mcb.8.8.3487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zhu X., Komiya H., Chirino A., Faham S., Fox G. M., Arakawa T., Hsu B. T., Rees D. C. Three-dimensional structures of acidic and basic fibroblast growth factors. Science. 1991 Jan 4;251(4989):90–93. doi: 10.1126/science.1702556. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES