Proteinase inhibitor I15, leech antistasin <p>Peptide proteinase inhibitors can be found as single domain proteins or as single or multiple domains within proteins; these are referred to as either simple or compound inhibitors, respectively. In many cases they are synthesised as part of a larger precursor protein, either as a prepropeptide or as an N-terminal domain associated with an inactive peptidase or zymogen. This domain prevents access of the substrate to the active site. Removal of the N-terminal inhibitor domain either by interaction with a second peptidase or by autocatalytic cleavage activates the zymogen. Other inhibitors interact direct with proteinases using a simple noncovalent lock and key mechanism; while yet others use a conformational change-based trapping mechanism that depends on their structural and thermodynamic properties. </p><p>This group of serine protease inhibitors belong to MEROPS inhibitor family I15, clan IO. They inhibit serine peptidases of the S1 family (<db_xref db="INTERPRO" dbkey="IPR001254"/>) [<cite idref="PUB00014133"/>] and are characterised by a well conserved pattern of cysteine residues. This is a family of leech anti-coagulants.</p><p>Antistasin is a 15kDa protein found in the salivary glands of <taxon tax_id="6410">Haementeria officinalis</taxon> (Mexican leech); it is an anticoagulant that functions byinhibiting factor Xa. The protein contains 119 residues, with an unusually high cysteine content (20 residues in all), and exhibits a 2-foldinternal repeated structure. Four isoforms of antistasin have beenidentified in leech salivary gland extracts; partial sequence analysis indicates that these isoforms differ only by 1 or 2 amino acid residues [<cite idref="PUB00010274"/>].</p><p>Ghilanten is an anticoagulant-antimetastatic protein of <taxon tax_id="6409">Haementeria ghilianii</taxon> (Amazon leech). Like antistasin, it contains 119 amino acids,with 20 cysteines, and a heparin-binding consensus motif at its C terminus.Arginine-34 is the residue involved in the active-site inhibition of trypsinand Factor Xa [<cite idref="PUB00010268"/>].</p><p>The 3D structure of antistasin has been determined to 1.9A resolution byX-ray crystallography [<cite idref="PUB00010320"/>]. The structure reveals a novel protein foldcomprising two similar domains, which can be divided into two similarly sized subdomains, with different relative orientations. Thus, the domainshapes differ, the N-terminal domain being wedge-shaped and the C-terminaldomain flat [<cite idref="PUB00010320"/>]. Docking studies suggest that it is differences in domainshape that enable the N-terminal domain to bind and inhibit factor Xa,rather than the C-terminal domain, despite very similar active sites. A putative exosite binding region is evident in the N-terminal domain(residues 15-17), which is likely to interact with a cluster of positivelycharged residues on the factor Xa surface (Arg222/Lys223/Lys224), explainingthe specificity and inhibitory potency of antistasin towards factor Xa.</p>