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  • The EMBO Journal (1997) 16, 5151 - 5161
  • doi:10.1093/emboj/16.17.5151

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

Risto Lapatto1,2, Ute Krengel1, Herman A. Schreuder3, Anita Arkema1, Bijtske de Boer1, Kor H. Kalk1, Wim G.J. Hol4, Peter D.J. Grootenhuis5, John W.M. Mulders5, Rein Dijkema5, Henri J.M. Theunissen5 and Bauke W. Dijkstra1,5

  1. Laboratory of Biophysical Chemistry and BIOSON Research Institute, Department of Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
  2. Present address: Children's Hospital and Institute of Biomedicine, University of Helsinki, 00014 Helsinki, Finland
  3. Present address: Hoechst Aktiengesellschaft, 65926 Frankfurt am Main, Germany
  4. Present address: Department of Biological Structure and Howard Hughes Medical Institute, University of Washington, Box 357742, Seattle, WA 98195, USA
  5. Scientific Development Group, N.V. Organon, PO Box 20, 5340 BH Oss, The Netherlands

Received 20 April 1997; Revised 12 June 1997

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 Å 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.

  • Keywords:

    • antistasin,
    • crystal structure,
    • factor Xa,
    • protease inhibitor,
    • thrombosis