Figure 1
Figure 1. Structure and function of Xase complex. (A) Schematic representation of Xase complex and mechanism of factor X activation. The major structural domains of cofactor VIIIa, its cognate protease FIXa, and substrate FX are labeled, and represented in the approximate positions they would occupy in the Xase complex. The long FX activation peptide is also indicated as a ribbon, pointing to the insertion of the Arg194(15)-Ile195(16) activation peptide bond in the active site of FIXa. (Numbers in parentheses refer to the standard chymotrypsinogen numbering system.) Gla indicates γ-carboxyglutamic–rich domain; EGF1/EGF2, epidermal growth factor–like domains 1 and 2; and SP, serine protease domain. (B) Three-dimensional model of human FVIIIa, highlighting the side chains of all novel missense mutations identified in the current investigation (light magenta spheres). The 5 FVIIIa domains are represented with their major secondary structure elements and color-coded as in panel A. For clarity, only selected residues are labeled, including the 4 exposed residues Arg64, Ala375, Gly494, and Asp2267 (boxed). The acidic a1 and a2 peptides are included only to indicate their locations on opposite poles of domain A2, as no appropriate templates are available for them. Selected side chains of hydrophobic residues in C1/C2 domains that would associate with the phospholipid membrane are in green. Some previously detected mutations of exposed residues are shown as yellow sticks; for clarity, only a few of these residues are labeled (see “Discussion” for details and references). Considering these previous analyses of FVIII mutants, residues important for cognate FIXa binding cluster to the right in the chosen orientation (eg, stretches Ser558-Gln565, Asp712/Lys713, and Glu1811-Lys1819), while those involved in FX recognition map to the left in this orientation (see, eg, the location of the acidic a1 peptide). This clear segregation of residues critical for FIXa/FX binding allows us to predict important roles for residues Pro64, Gly494, and Asp2267 in substrate binding and presentation to the FVIIIapi•FIXa (Xase) complex.

Structure and function of Xase complex. (A) Schematic representation of Xase complex and mechanism of factor X activation. The major structural domains of cofactor VIIIa, its cognate protease FIXa, and substrate FX are labeled, and represented in the approximate positions they would occupy in the Xase complex. The long FX activation peptide is also indicated as a ribbon, pointing to the insertion of the Arg194(15)-Ile195(16) activation peptide bond in the active site of FIXa. (Numbers in parentheses refer to the standard chymotrypsinogen numbering system.) Gla indicates γ-carboxyglutamic–rich domain; EGF1/EGF2, epidermal growth factor–like domains 1 and 2; and SP, serine protease domain. (B) Three-dimensional model of human FVIIIa, highlighting the side chains of all novel missense mutations identified in the current investigation (light magenta spheres). The 5 FVIIIa domains are represented with their major secondary structure elements and color-coded as in panel A. For clarity, only selected residues are labeled, including the 4 exposed residues Arg64, Ala375, Gly494, and Asp2267 (boxed). The acidic a1 and a2 peptides are included only to indicate their locations on opposite poles of domain A2, as no appropriate templates are available for them. Selected side chains of hydrophobic residues in C1/C2 domains that would associate with the phospholipid membrane are in green. Some previously detected mutations of exposed residues are shown as yellow sticks; for clarity, only a few of these residues are labeled (see “Discussion” for details and references). Considering these previous analyses of FVIII mutants, residues important for cognate FIXa binding cluster to the right in the chosen orientation (eg, stretches Ser558-Gln565, Asp712/Lys713, and Glu1811-Lys1819), while those involved in FX recognition map to the left in this orientation (see, eg, the location of the acidic a1 peptide). This clear segregation of residues critical for FIXa/FX binding allows us to predict important roles for residues Pro64, Gly494, and Asp2267 in substrate binding and presentation to the FVIIIapi•FIXa (Xase) complex.

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