X-ray structure of antistasin at 1.9 angstrom resolution and its modelled complex with blood coagulation factor Xa

Abstract

The three-dimensional structure of antistasin, a potent inhibitor of blood coagulation factor Xa, from the Mexican leech Haementeria officinalis was determined at 1.9 Angstrom resolution by X-ray crystallography, The structure reveals a novel protein fold composed of two homologous domains, each resembling the structure of hirustasin, a related 55-residue protease inhibitor, However, hirustasin has a different overall shape than the individual antistasin domains, it contains four rather than two beta-strands, and does not inhibit factor Xa, The two antistasin domains can be subdivided into two similarly sized subdomains with different relative orientations, Consequently, the domain shapes are different, the N-terminal domain being wedge-shaped and the C-terminal domain flat, Docking studies suggest that differences in domain shape enable the N-terminal, but not C-terminal, domain of antistasin to bind and inhibit factor Xa, even though both have a very similar reactive site, Furthermore, a putative exosite binding region could be defined in the N-terminal domain of antistasin, comprising residues 15-17, which is likely to interact with a cluster of positively charged residues on the factor Xa surface (Arg222/Lys223/Lys224). This exosite binding region explains the specificity and inhibitory potency of antistasin towards factor Xa, In the C-terminal domain of antistasin, these exosite interactions are prevented due to the different overall shape of this domain