Inherent flexibility in a potent inhibitor of blood coagulation, recombinant nematode anticoagulant protein c2

Nematode anticoagulant proteins (NAPs) from the hematophagous nematode Ancy lostoma caninum inhibit blood coagulation with picomolar inhibition constan ts, and have been targeted as novel pharmaceutical agents. NAPS and NAP6 in hibit factor Xa by binding to its active site, whereas NAPc2 binds to facto r Xa at a different, as yet unidentified, site and the resultant binary com plex inhibits the tissue factor-factor Wa complex. We have undertaken NMR s tudies of NAPc2, including the calculation of a solution structure, and fou nd that the protein is folded, with five disulfide bonds, but is extremely flexible, especially in the acidic loop. The Ha secondary shifts and (3)J(H NH alpha) coupling constants indicate the presence of some beta structure a nd a short helix, but the intervening loops are highly conformationally het erogeneous. Heteronuclear NOE measurements showed the presence of large amp litude motions on a subnanosecond timescale at the N-terminus and C-terminu s and in the substrate-binding loop, indicating that the conformational het erogeneity observed in the NMR structures is due to flexibility of the poly peptide chain in these regions. Flexibility may well be an important factor in the physiological function of NAPc2, because it must interact with othe r proteins in the inhibition of blood coagulation. We suggest that this inh ibitor is likely to become structured on binding to factor Xa, because the inhibition of the tissue factor-factor VIIa complex requires both NAPc2 and factor Xa.