Protease and EGF1 domains of factor IXa play distinct roles in binding to factor VIIIa. Importance of helix 330 (helix 162 in chymotrypsin) of protease domain of factor IXa in its interaction with factor VIIIa.
Previous studies revealed that cleavage at Arg-318-Ser-319 in the protease domain autolysis loop of factor IXa results in its diminished binding to factor VIIIa. Now, we have investigated the importance of adjacent surface-exposed helix 330-338 (162-170 in chymotrypsin numbering) of IXa in its interaction with VIIIa. IXWT, eight point mutants mostly based on hemophilia B patients, and a replacement mutant (IXhelixVII in which helix 330-338 is replaced by that of factor VII) were expressed, purified, and characterized. Each mutant was activated normally by VIIa-tissue factor-Ca2+ or XIa-Ca2+. However, in both the presence and absence of phospholipid, interaction of each activated mutant with VIIIa was impaired. The role of IXa EGF1 domain in binding to VIIIa was also examined. Two mutants (IXQ50P and IXPCEGF1, in which EGF1 domain is replaced by that of protein C) were used. Strikingly, interactions of the activated EGF1 mutants with VIIIa were impaired only in the presence of phospholipid. We conclude that helix 330 in IXa provides a critical binding site for VIIIa and that the EGF1 domain in this context primarily serves to correctly position the protease domain above the phospholipid surface for optimal interaction with VIIIa.
Mathur
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Departments of Medicine, Pathology, and Biochemistry, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA.
Bajaj
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D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005164:Factor IX / Q000235:genetics; D015949:Factor IXa / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
D015944:Factor VIIIa; D005164:Factor IX; D002918:Chymotrypsin; D015949:Factor IXa