Conformation of heparin pentasaccharide bound to antithrombin III

Abstract

The interaction, in aqueous solution, of the synthetic pentasaccharide AGA*IA(M) (GlcN,6-SO(3)alpha 1-4GlcA beta 1-4GlcN,3,6-SO(3)alpha 1-4IdoA,2-SO(3)alpha 1-4GlcN,6-SO(3)alpha OMe; where GlcN,6-SO(3) is 2-deoxy-2-sulphamino-alpha-D-glucopyranosyl 6-sulphate, IdoA is l-iduronic acid and IdoA2-SO(3) is L-iduronic acid 2-sulphate), which exactly reproduces the structure of the specific binding sequence of heparin and heparan sulphate for antithrombin III, has been studied by NMR. In the presence of antithrombin there were marked changes in the chemical shifts and nuclear Overhauser effects (NOEs), compared with the free state. On the basis of the optimized geometry of the pentasaccharide the transferred NOEs were interpreted with full relaxation and conformational exchange matrix analysis. An analysis of the three-dimensional structures of the pentasaccharide in the free state, and in the complex, revealed the binding to be accompanied by dihedral angle variation at the A-G and I-A(M) (where G, I, A and A(M) are beta-d-glucuronic acid, 2-O-sulphated alpha-L-iduronic acid, N,6-O-sulphated alpha-D-glucosamine and the alpha-methyl-glycoside of A respectively) glycosidic linkages. Evidence is also provided that the protein drives the conformation of the 2-O-sulphated iduronic acid residue towards the skewed (2)S(0) form.

Antithrombin-binding Octasaccharides and Role of Extensions of the Active Pentasaccharide Sequence in the Specificity and Strength of Interaction

“…A significant enhancement of H5-H2/H5-H4 NOE ratio of the I residue was observed for both OCTA-2 and OCTA-4. This indicates that the conformation of such moiety is driven toward the 2 S O form by the presence of AT, as observed in all AGA*IAcontaining oligosaccharides so far described (10,12,15). In contrast, the IЉ residue of OCTA-2 does not show an H5-H2 tr-NOE signal, indicating that IЉ maintains its 1 C 4 conformation in the presence of AT.…”

“…Despite the strong evidence accumulated on the specificity of heparin-AT binding, (3,10,15), recent studies suggested other possible assemblies between the negatively charged heparin chains and AT (38,39). In these latter studies, the possibility has been considered that sulfated residues in heparin sequences, different from that of the AT-binding site, may activate AT through nonspecific interactions.…”

“…A ligand/protein ratio of 3.5:1 was set for OCTA-2, -3, and -4, and a ratio of 5:1 was set for OCTA-1. The correlation time of AT was considered as isotropic and estimated from published data ( AT , 46 ns) (15), whereas an average correlation time value characteristic of oligosaccarides having similar structure was used for octasaccharides ( octasaccharide , 0.9 ns) (28).…”

“…The possibility of a shift along the AT D-helix for sequences longer than pentasaccharide was taken into consideration (11). Independent crystallographic and NMR studies on the structure of complexes of AT with AGA*IA and AGA*IA-containing oligosaccharides suggested that the position of the pentasaccharide in the protein binding region is unique (10,(12)(13)(14)(15). These studies provided information on both the ring conformation of the monosaccharide residues and the geometry of the glycosidic linkages of the AT-bound pentasaccharide.…”

“…In particular, it was shown that the 2-O-sulfated iduronic acid residue in the pentasaccharide, which in the free state in water solution is in equilibrium between two equienergetic conformations ( 1 C 4 , and 2 S O ) (16), adopts the 2 S O conformation when AGA*IA is bound to AT. Shifting toward this conformation, facilitated by the presence of the 2-OSO 3 group, enhances the contacts between the AGA*IA and basic amino acid residues in the AT binding region (15).…”