抄録
Hereditary deficiency of factor IXa (fIXa), a key enzyme in blood coagulation, causes haemophilia B, an X-linked severe bleeding disorder occurring in approximately 1 in 30, 000 males. About 500 different mutations, more than for any other human gene, have been described for fIX. The X-ray crystal structure of full-length porcine fIXaβ determined in the absence of calcium reveals a fXa-like catalytic module, with D-Phe-Pro-Argchloromethyl-ketone (PPACK) covalently bound to an active site of thrombin-like geometry. The entrance frame to the specificity pocket, however, displays a considerable trembling motion imposed by the high-energy conformation of residue Glu388 (C219), which is strictly conserved in fIX species but replaced by glycines in all other serine proteinases of trypsin-like specificity. Dynamic simulations suggest a cryptic pocket in unliganded fIXaβ and the requirement for its opening upon substrate binding, in agreement with the very low activity of free fIXaβ towards chromogenic substrates. The two EGF-like domains interact at an angle of 110°, stabilized by a strictly conserved and fIX specific inter-domain salt bridge. The Gla domain, only defined for its hydrophobic stack, has been modeled in its calcium form using prothrombin fragment 1. Due to the inter-EGF bend, the distance from the active site residues to the expected membrane plane is almost 90Å, in fair agreement with results obtained from fluorescence energy transfer experiments. Most natural mutations, which affect conserved fIX-specific surface residues, map to the concave surface strip of the bent molecule, probably indicating its involvement in intrinsic fXase complex formation. In the membrane-bound complex, fIXaβ and its substrate fX seem to envelope parts of fVIIIa from opposite sides with their catalytic modules pushed together, so that the opening of fIXa's active site is enforced. This model could explain the tremendous activity enhancement of fIXa upon complex formation.
