Rios-Steiner Jorge L, Murakami Mário T, Tulinsky Alexander, Arni Raghuvir K
Department of Chemistry, Michigan State University, East Lansing, MI 48824-1322, USA.
J Mol Biol. 2007 Aug 17;371(3):774-86. doi: 10.1016/j.jmb.2007.05.042. Epub 2007 May 18.
Hookworms are hematophagous nematodes capable of growth, development and subsistence in living host systems such as humans and other mammals. Approximately one billion, or one in six, people worldwide are infected by hookworms causing gastrointestinal blood loss and iron deficiency anemia. The hematophagous hookworm Ancylostoma caninum produces a family of small, disulfide-linked protein anticoagulants (75-84 amino acid residues). One of these nematode anticoagulant proteins, NAP5, inhibits the amidolytic activity of factor Xa (fXa) with K(i)=43 pM, and is the most potent natural fXa inhibitor identified thus far. The crystal structure of NAP5 bound at the active site of gamma-carboxyglutamic acid domainless factor Xa (des-fXa) has been determined at 3.1 A resolution, which indicates that Asp189 (fXa, S1 subsite) binds to Arg40 (NAP5, P1 site) in a mode similar to that of the BPTI/trypsin interaction. However, the hydroxyl group of Ser39 of NAP5 additionally forms a hydrogen bond (2.5 A) with His57 NE2 of the catalytic triad, replacing the hydrogen bond of Ser195 OG to the latter in the native structure, resulting in an interaction that has not been observed before. Furthermore, the C-terminal extension of NAP5 surprisingly interacts with the fXa exosite of a symmetry-equivalent molecule forming a short intermolecular beta-strand as observed in the structure of the NAPc2/fXa complex. This indicates that NAP5 can bind to fXa at the active site, or the exosite, and to fX at the exosite. However, unlike NAPc2, NAP5 does not inhibit fVIIa of the fVIIa/TF complex.
钩虫是一种吸血性线虫，能够在人类和其他哺乳动物等活体宿主系统中生长、发育和生存。全球约有10亿人，即六分之一的人口感染钩虫，导致胃肠道失血和缺铁性贫血。吸血性钩虫犬钩口线虫产生一族小的、通过二硫键连接的蛋白质抗凝剂（75 - 84个氨基酸残基）。这些线虫抗凝蛋白之一，NAP5，以K(i)=43 pM的亲和力抑制因子Xa（fXa）的酰胺水解活性，是迄今为止鉴定出的最有效的天然fXa抑制剂。已确定NAP5与无γ-羧基谷氨酸结构域的因子Xa（des - fXa）活性位点结合的晶体结构，分辨率为3.1 Å，这表明Asp189（fXa，S1亚位点）以类似于BPTI/胰蛋白酶相互作用的模式与Arg40（NAP5，P1位点）结合。然而，NAP5的Ser39羟基额外与催化三联体的His57 NE2形成氢键（2.5 Å），取代了天然结构中Ser195 OG与后者的氢键，导致一种前所未有的相互作用。此外，NAP5的C末端延伸部分令人惊讶地与对称等效分子的fXa外位点相互作用，形成短的分子间β链，这在NAPc2/fXa复合物的结构中也有观察到。这表明NAP5可以在活性位点或外位点与fXa结合，并在外位点与fX结合。然而，与NAPc2不同，NAP5不抑制fVIIa/TF复合物中的fVIIa。
