Role of N- and C-terminal amino acids in antithrombin binding to pentasaccharide.
We have used a monoclonal antibody-based binding procedure to determine the dissociation constants of the interactions between the essential antithrombin-binding pentasaccharide and a series of 13 distinct N- and C-terminal antithrombin substitution mutation variants with defective binding interaction with heparin. The reduction in binding affinity of the pentasaccharide with the N-terminal variants (with substitution mutations Pro-41-->Leu, Arg-47-->Cys and His, Leu-99-->Val and Phe, Gln-118-->Pro, Arg-129-->Gln) compared to normal antithrombin, Kd 200 nM, ranged from 15-984-fold and was generally less than 150-fold. Reduced binding affinity is assumed to arise mostly by perturbation, direct or indirect, of the initial contact of pentasaccharide with basic residues of antithrombin. Surprisingly, the binding interaction of the pentasaccharide with the C-terminal variants (with substitution mutations in or near strand 1C/4B, Phe-402-->Leu, Cys, and Ser, Ala-404-->Thr, Pro-407-->Thr, Pro-429-->Leu) was more uniformly and yet substantially (135-482-fold) decreased, despite the spatial separation between the site of mutation and the proposed primary contact site of the pentasaccharide. These results demonstrate that strand 1C/4B region integrity is required for optimum interaction with the pentasaccharide, suggesting its involvement in transmission of the induced conformation change required for high affinity binding.
Mille
B
B
Department of Haematology, Charing Cross and Westminister Medical School, London, United Kingdom.
Watton
J
J
Barrowcliffe
T W
TW
Mani
J C
JC
Lane
D A
DA
D000596:Amino Acids / Q000378:metabolism*; D000991:Antithrombins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D006493:Heparin / Q000378:metabolism; D006801:Humans; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism*
D000596:Amino Acids; D000991:Antithrombins; D009844:Oligosaccharides; D006493:Heparin