Inherent flexibility in a potent inhibitor of blood coagulation, recombinant nematode anticoagulant protein c2.
Nematode anticoagulant proteins (NAPs) from the hematophagous nematode Ancylostoma caninum inhibit blood coagulation with picomolar inhibition constants, and have been targeted as novel pharmaceutical agents. NAP5 and NAP6 inhibit factor Xa by binding to its active site, whereas NAPc2 binds to factor Xa at a different, as yet unidentified, site and the resultant binary complex inhibits the tissue factor-factor VIIa complex. We have undertaken NMR studies of NAPc2, including the calculation of a solution structure, and found that the protein is folded, with five disulfide bonds, but is extremely flexible, especially in the acidic loop. The Halpha secondary shifts and 3JHNHalpha coupling constants indicate the presence of some beta structure and a short helix, but the intervening loops are highly conformationally heterogeneous. Heteronuclear NOE measurements showed the presence of large amplitude motions on a subnanosecond timescale at the N-terminus and C-terminus and in the substrate-binding loop, indicating that the conformational heterogeneity observed in the NMR structures is due to flexibility of the polypeptide chain in these regions. Flexibility may well be an important factor in the physiological function of NAPc2, because it must interact with other proteins in the inhibition of blood coagulation. We suggest that this inhibitor 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.
Duggan
B M
BM
Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Dyson
H J
HJ
Wright
P E
PE
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry*; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D015942:Factor VIIa / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry; D015801:Helminth Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009348:Nematoda; D010843:Pichia; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment
D000925:Anticoagulants; D004220:Disulfides; D015801:Helminth Proteins; D011994:Recombinant Proteins; C400226:anti-coagulant protein C2, Ancylostoma caninum; D015942:Factor VIIa; D015951:Factor Xa