Active Conformations of Glycosaminoglycans. NMR Determination of the Conformation of Heparin Sequences Complexed with Antithrombin and Fibroblast Growth Factors in Solution

 

 Buy Article Permissions and Reprints

ABSTRACT

Binding to proteins usually induces perturbation of nuclear magnetic resonances of ligand molecules. Using sensitive nuclear magnetic resonance (NMR) spectroscopy techniques, these perturbations have been measured for heparin oligosaccharides in aqueous solution in the presence of proteins and the NMR data have been used to characterize the three-dimensional (3D) structure of the oligosaccharides in the bound state. The pentasaccharide corresponding to the active site of heparin/heparan sulfate for antithrombin (AT) adopts in the complex with the protein a conformation different from that in the absence of the protein. A notable difference involves the 2-O-sulfated iduronic acid (IdoA2S) residue, which is driven to adopt an exclusively skew-boat

@affil2:²S₀ form in the complex. In addition, complexing induces a change in the geometry around the glycosidic linkage between the nonreducing end glucosamine and the adjacent glucuronic acid residue as compared with the free state. NMR and molecular modeling data also indicate that the 2-O-sulfate group in the IdoA2S residue is not directly involved in binding to AT. This suggests that its role is mainly that of affecting the conformational equilibrium of this residue, leading to a 3D structure of pentasaccharide in the bound state that meets the stereochemical requirements of the receptor and results in high-affinity binding to the protein. On the other hand, NMR studies of heparin tetrasaccharides in the presence of fibroblast growth factors FGF-1 and FGF-2 indicate that FGF binding stabilizes the

@affil1:¹C₄ conformation of the IdoA2S residue directly involved in binding. These studies also confirm the crucial role of the 6-O-sulfate group on at least one glucosamine residue in the formation of the complex with FGF-1 but not with FGF-2.

REFERENCES

• 1 Casu B, Lindahl U. Structure and biological interactions of heparin and heparan sulfate.  Adv Carbohydr Chem Biochem . 2001;  57 159-206
  PubMedGoogle Scholar
• 2 Capila I, Linhard R J. Heparin-protein interactions.  Angew Chem Int Ed . 2002;  41 390-412
  PubMedGoogle Scholar
• 3 Casu B, Ferro D R, Ragazzi M, Torri G. Conformation of iduronic acid-containing glycosaminoglycans. In: Scott JE, ed. Dermatan Sulphate Proteoglycans Chemistry, Biology, Chemical Pathology London: Portland Press 1993: 41-53
  Google Scholar
• 4 Ragazzi M, Ferro D R, Perly B. Conformation of the pentasaccharide corresponding to the binding site of heparin for antithrombin III.  Carbohydr Res . 1987;  165 c1-c5
  CrossrefPubMedGoogle Scholar
• 5 Mikhailov D, Linhardt R J, Mayo K H. NMR solution conformation of heparin-derived hexasaccharide.  Biochem J . 1997;  328 51-61
  PubMedGoogle Scholar
• 6 Casu B, Choay J, Ferro D R. Controversial glycosaminoglycan conformations.  Nature . 1986;  322 215-216
  PubMedGoogle Scholar
• 7 Ferro D R, Provasoli A, Ragazzi M. Evidence for conformational equilibrium of the sulfated l-iduronate residue in heparin and in synthetic heparin mono- and oligosaccharides: NMR and force-field studies.  J Am Chem Soc . 1986;  108 6773-6778
  CrossrefPubMedGoogle Scholar
• 8 Casu B, Petitou M, Provasoli M, SinaĀ P. Conformational flexibility, a new concept for explaining protein binding and associated biological properties of iduronic acid-containing glycosaminoglycans.  Trends Biochem Sci . 1998;  13 221-225
  PubMedGoogle Scholar
• 9 Ragazzi M, Ferro D M, Provasoli M. A force-field study of the conformational characteristics of the iduronate rings.  J Comp Chem . 1986;  7 105-112
  PubMedGoogle Scholar
• 10 Mulloy B, Forster M, Jones C, Davies D B. NMR molecular modeling studies of the solution conformation of heparin.  Biochem J . 1993;  293 849-858
  PubMedGoogle Scholar
• 11 Jin L, Abrahams J P, Skinner R. Activation of antithrombin by heparin.  Proc Natl Acad Sci USA . 1997;  94 14683-14688
  CrossrefPubMedGoogle Scholar
• 12 Faham S, Hileman R E, Fromm J R. Heparin structure and interaction with basic fibroblast growth factor.  Science . 1996;  271 1116-1120
  PubMedGoogle Scholar
• 13 Poveda A, Barbero J J. NMR studies of carbohydrate-protein interactions in solution.  Chem Soc Rev . 1998;  27 133-143
  PubMedGoogle Scholar
• 14 Hricovíni M, Nieto P N, Torri G. NMR of sulfated oligo and polysaccharides. In: Barbero JJ, Peters T, eds. NMR of Glycoconjugates (in press) Weinheim, Germany: Wiley-VCH 2002
  Google Scholar
• 15 Ragazzi M, Ferro D R, Perly B. Conformation of the pentasaccharide corresponding to the binding site of heparin for antithrombin III.  Carbohydr Res . 1990;  15 169-185
  PubMedGoogle Scholar
• 16 Hricovíni M, Torri G. Dynamics in aqueous solution of the pentasaccharide corresponding to the binding site of heparin for antithrombin III studied by NMR relaxation measurements.  Carbohydr Res . 1995;  268 159-175
  CrossrefPubMedGoogle Scholar
• 17 Hricovíni M, Guerrini M, Bisio A. Conformation of heparin pentasaccharide bound to antithrombin III.  Biochem J . 2001;  359 265-272
  CrossrefPubMedGoogle Scholar
• 18 Hricovíni M, Guerrini M, Bisio A. Structure of heparin-derived tetrasaccharide complexed to the plasma protein antithrombin derived from NOEs, J-couplings and chemical shifts.  Eur J Biochem . 1999;  261 789-801
  CrossrefPubMedGoogle Scholar
• 19 Lei P S, Duchaussoy P, Sizun P. Containing heparin pentasaccharide to probe the conformation of the antithrombin III binding sequence.  Bioorg Med Chem . 1998;  6 1337-1346
  PubMedGoogle Scholar
• 20 Herr A B, Ornitz D M, Sasisekharan R, Venkataraman G, Waksman G. Heparin-induced self association of fibroblast growth factor-2. Evidence for two oligomerization processes.  J Biol Chem . 1997;  272 16382-16389
  CrossrefPubMedGoogle Scholar
• 21 Waksman G, Herr A B. New insights into heparin-induced FGF oligomerization.  Nat Struct Biol . 1998;  5 527-530
  CrossrefPubMedGoogle Scholar
• 22 Maccarana M, Casu B, Lindahl U. Minimal sequence in heparin-heparan sulfate required for binding of basic fibroblast growth factor.  J Biol Chem . 1993;  268 23898-23905
  PubMedGoogle Scholar
• 23 DiGabriele A D, Lax I, Chen D I. Structure of a heparin-linked biologically active dimer of fibroblast growth factor.  Nature . 1998;  393 812-817
  CrossrefPubMedGoogle Scholar
• 24 Ornitz D M, Herr A B, Nilsson M. FGF binding and FGF receptor activation by synthetic heparan-derived di- and trisaccharides.  Science . 1995;  268 432-436
  PubMedGoogle Scholar
• 25 Poletti L, Lay L, Fleischer M. Rational approach to heparin-related fragments. Synthesis of differently sulfated tetrasaccharides as potential ligands towards fibroblast growth factors.  Eur J Org Chem . 2001;  14 2727-2734
  PubMedGoogle Scholar
• 26 Guerrini M, Agulles T, Bisio A. Minimal heparin/heparan sulfate sequences for binding to fibroblast growth factor-1.  Biochem Biophys Res Commun . 2002;  292 222-230
  CrossrefPubMedGoogle Scholar
• 27 Kreuger J, Salmivirta M, Sturiale L, Giménez-Gallego G, Lindahl U. Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2.  J Biol Chem . 2001;  276 30744-30752
  CrossrefPubMedGoogle Scholar

1 *In collaboration with L. Lay and L. Poletti, University of Milan.