The interaction, in aqueous solution, of the synthetic pentasaccharide AGA∗IAM (GlcN,6-SO3α1–4GlcAβ1–4GlcN,3,6-SO3α1–4IdoA,2-SO3α1–4GlcN,6-SO3αOMe; where GlcN,6-SO3 is 2-deoxy-2-sulphamino-α-d-glucopyranosyl 6-sulphate, IdoA is l-iduronic acid and IdoA2-SO3 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–AM (where G, I, A and AM are β-d-glucuronic acid, 2-O-sulphated α-l-iduronic acid, N,6-O-sulphated α-d-glucosamine and the α-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 2S0 form.
- NMR spectroscopy
- protein–carbohydrate interaction
- solution structure
Abbreviations used: 3D, three-dimensional; AT, antithrombin III; IdoA, l-iduronic acid; IdoA2-SO3, l-iduronic acid 2-sulphate; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; I, 2-O-sulphated α-l-iduronic acid; A, N,6-O-sulphated α-d-glucosamine; G, β-d-glucuronic acid; A∗, N,3,6-O-sulphated α-d-glucosamine; AM, α-methyl-glycoside of A.
- The Biochemical Society, London ©2001