Structure–Function Relations of Antithrombin III–Heparin Interactions as Assessed by Biophysical and Biological Assays and Molecular Modeling of Peptide–Pentasaccharide-Docked Complexes

The serine proteinase inhibitor antithrombin III (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated glycosaminoglycans, such as heparin. Peptide K121–A134, based on the sequence of ATIII in the D helix region, was previously shown by us (Tyler-Crosset al., Protein Sci.3, 620–627, 1994) to encompass part (or all) of the purported high-affinity heparin binding region of ATIII. A series of peptide analogs has now been prepared whose sequences are identical to K121–A134except that single cationic residues of K121–A134have been successively replaced with Ala. In one case, the Arg residue of the reference peptide corresponding to R129of ATIII has been replaced by Gln (R129ΔQ peptide), thus mimicking the naturally occurring mutant protein, ATIII Geneva. The heparin affinity of all peptides was quantitated by isothermal titration calorimetry and by peptide/ATIII competition binding assays. Replacement of any single cationic residue with Ala had a deleterious effect on heparin binding. The greatest reduction in heparin affinity (more than 30-fold) was observed with the R129ΔQ peptide (KD= 1.5 ± 0.06 μMvs 51 ± 2 nMfor the reference peptide, K121–A134). Furthermore, each of the Ala-replacement peptides was a less-effective inhibitor of ATIII–heparin complex formation than the reference peptide. The poorest inhibitor was the R129ΔQ peptide which showed nearly 30% decrease in inhibition potency (60% inhibition at 100 μMpeptide vs 90% inhibition with the reference peptide). The relative heparin affinities of the peptides measured by biological assay were the same as determined by titration calorimetry. Consequently, we modeled the complexes formed between the pentasaccharide unit structure and the R129ΔQ peptide or the reference peptide, K121–A134. In the “docked” complex, the assumed conformation of K121–A134permitted juxtaposition of the cationic residues of the peptide with functional anionic groups of the pentasaccharide known to be involved in binding. A docked complex could also be formed between the R129ΔQ peptide and the pentasaccharide, but misalignment of critical peptide and saccharide functional groups was observed. The structure of the R129ΔQ–pentasaccharide complex was highly irregular because F123and Y131were completely surface exposed, likely yielding an unfavorable structure in aqueous solution. The observations from molecular modeling allow us to suggest that ATIII Geneva displays decreased heparin binding affinity due to its inability to form a productive binding complex in which essential electrostatic contacts are made between suitably juxtaposed saccharide anionic functional groups and cationic amino acid side chains.