RESUMO
NAPc2, an anticoagulant protein from the hematophagous nematode Ancylostoma caninum evaluated in phase-II/IIa clinical trials, inhibits the extrinsic blood coagulation pathway by a two step mechanism, initially interacting with the hitherto uncharacterized factor Xa exosite involved in macromolecular recognition and subsequently inhibiting factor VIIa (K(i)=8.4 pM) of the factor VIIa/tissue factor complex. NAPc2 is highly flexible, becoming partially ordered and undergoing significant structural changes in the C terminus upon binding to the factor Xa exosite. In the crystal structure of the ternary factor Xa/NAPc2/selectide complex, the binding interface consists of an intermolecular antiparallel beta-sheet formed by the segment of the polypeptide chain consisting of residues 74-80 of NAPc2 with the residues 86-93 of factor Xa that is additional maintained by contacts between the short helical segment (residues 67-73) and a turn (residues 26-29) of NAPc2 with the short C-terminal helix of factor Xa (residues 233-243). This exosite is physiologically highly relevant for the recognition and inhibition of factor X/Xa by macromolecular substrates and provides a structural motif for the development of a new class of inhibitors for the treatment of deep vein thrombosis and angioplasty.
Assuntos
Ancylostoma/química , Fator Xa/química , Proteínas de Helminto/química , Animais , Anticoagulantes/farmacologia , Sítios de Ligação , Bovinos , Fator VIIa/química , Fator VIIa/metabolismo , Fator Xa/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Tromboplastina/química , Tromboplastina/metabolismoRESUMO
Thrombovascular diseases result from imbalanced haemostasis and comprise important health problems in the aging population worldwide. The activity of enzymes pertaining to the coagulation cascade of mammalians exhibit several control mechanisms in order to maintain a proper balance between bleeding and thrombosis. For instance, human coagulation serine proteases carrying a F225 or Y225 are allosteric modulated by the binding of Na+ in a water-filled channel connected to the primary specificity pocket (S1 subsite) of these enzymes. We have characterized the structure, topography and lipophilicity of this channel in the ligand-free fast (sodium-bound) and slow (sodium-free) forms of thrombin, in the sole available structure of activated protein C and in several structures of the coagulation factors VIIa, IXa and Xa, differing in the nature of the bound inhibitor and in the occupancy of exosite-I as well as the Ca2+ and Na+ binding sites. Opposite to thrombin, the aqueous channels in all other coagulation enzymes sheltering a Na+ binding site do not have an aperture on the enzyme surface opposite to the S1 subsite entrance. In these enzymes, the lack of the three-residue insertion in loop 1 (183-189) as found in thrombin allied to compensatory mutations in the positions 187-185 and 222 effects a constriction in the water-filled channel that ends up by segregating the ion binding site from the S1 subsite. We also disclosed major topographical changes on the thrombin's surface upon sodium release and transition to the slow form that culminate in the narrowing of the S1 subsite entrance and, strikingly, in the loss of communication between the primary specificity pocket and the exosite-I. Such observation is in accordance with existing experimental data demonstrating thermodynamic linkage between these distant regions on the thrombin surface. Conformational changes in F34, L40, R73 and T74 were the main responsible for this effect. A path by which these changes in the vicinity of exosite-I could be transmitted to the S1 subsite and, consequently, to the sodium binding site is proposed.