The Extended Interactions and Gla Domain of Blood Coagulation Factor Xa

Abstract

The serine protease factor Xa (FXa) is inhibited by ecotin with picomolar affinity. The structure of the tetrameric complex of ecotin variant M84R (M84R) with FXa has been determined to 2.8 A. Substrate directed induced fit of the binding interactions at the S2 and S4 pockets modulates the discrimination of the protease. Specifically, the Tyr at position 99 of FXa changes its conformation with respect to incoming ligand, changing the size of the S2 and S4 pockets. The role of residue 192 in substrate and inhibitor recognition is also examined. Gln 192 from FXa forms a hydrogen bond with the P2 carbonyl group of ecotin. This confirms previous biochemical and structural analyses on thrombin and activated protein C, which suggested that residue 192 may play a more general role in mediating the interactions between coagulation proteases and their inhibitors. The structure of ecotin M84R-FXa (M84R-FXa) also reveals the structure of the Gla domain in the presence of Mg(2+). The first 11 residues of the domain assume a novel conformation and likely represent an intermediate folding state of the domain.

The Serine Protease Domain of MASP-3: Enzymatic Properties and Crystal Structure in Complex with Ecotin

“…These include two canonical central motifs: the main-chain nitrogens of G643( 193 ) and S645( 195 ), defining the ‘oxyanion hole’, stabilize the ecotin M84 carbonyl; S82 of ecotin is stabilized by two main-chain H-bonds with G668( 216 ) of MASP-3. The MASP-3 Y531( 99 ) side chain interacts with the ecotin H53-R54 main-chain, as also observed in the case of fXa [52]. The interactions mediated by the ecotin loops 50–53 and 79–86 also share similar features with those mediated by inhibitors of the Pacifastin family, as shown in the case of the SGMI-2/MASP-2 complex (Fig.…”

“…Since the crystal structure of MASP-3 alone is not available, the free and ecotin-bound conformations cannot be compared. However, ecotin-induced conformational changes have been observed previously in the case of thrombin and factor Xa [52], and significant conformational changes were also observed in the catalytic fragment of MASP-2 in complex with SGMI-2 [57]. Evidence of such a change may be inferred from the comparison of the position and orientation of Tyr531(99) in MASP-3 and its homologues.…”

“…7A). In fXa, this residue is significantly displaced by its close interaction with loop 50 of ecotin [52]. Its conformation in ecotin-bound MASP-3 is far more similar to that in the ecotin-bound fXa (not shown) or in the SGMI-bound MASP-1 and -2 than in the free forms of MASP-1, MASP-2, C1r, C1s and fXa (Fig.…”

“…A major objective was indeed to obtain complexes suitable for crystallographic studies, since initial attempts to obtain good crystals of different MASP-3 fragments had failed. Ecotin has been previously described as a molecular tool to assist the crystallization of proteases [38], since the X-ray structures of complexes between ecotin (or variants) and various proteases have been reported, including trypsin [48], collagenase [49], thrombin [50], granzyme B [51], coagulation factors Xa [52] and XIa [38].…”

Determinants of Specificity of Factor Xa Interaction with its Physiological Inhibitors

“…In addition to TFPI, a number of non-physiological, but naturally occurring peptide inhibitors specific for fXa have recently been identified which inhibit the protease by a similar slow and tight-binding inhibition mechanism [71][72][73][74]. The interaction of a recombinant form of such an inhibitor possessing potent antithrombotic properties, derived from the soft tick Ornithodoros moubata (tick anticoagulant peptide, TAP, I52.001), with fXa has been extensively studied [72].…”