The tissue factor region that interacts with substrates factor IX and Factor X.
The enzymatic activity of coagulation factor VIIa is controlled by its cellular cofactor tissue factor (TF). TF binds factor VIIa with high affinity and, in addition, participates in substrate interaction through its C-terminal fibronectin type III domain. We analyzed surface-exposed residues in the C-terminal TF domain to more fully determine the area on TF important for substrate activation. Soluble TF (sTF) mutants were expressed in E. coli, and their ability to support factor VIIa-dependent substrate activation was measured in the presence of phospholipid vesicles or SW-13 cell membranes. The results showed that factor IX and factor X interacted with the same TF region located proximal to the putative phospholipid surface. According to the degree of activity loss of the sTF mutants, this TF region can be divided into a main region (residues Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, Tyr185) forming a solvent-exposed patch of 488 A(2) and an extended region which comprises an additional 7-8 residues, including the distally positioned Asn199, Arg200, and Asp204. Some of the identified TF residues, such as Trp158 and those within the loop Lys159-Lys165, are near the factor VIIa gamma-carboxyglutamic acid (Gla) domain, suggesting that the factor VIIa Gla-domain may also participate in substrate interaction. Moreover, the surface identified as important for substrate interaction carries a net positive charge, suggesting that charge interactions may significantly contribute to TF-substrate binding. The calculated surface-exposed area of this substrate interaction region is about 1100 A(2), which is approximately half the size of the TF area that is in contact with factor VIIa. Therefore, a substantial portion of the TF surface (3000 A(2)) is engaged in protein-protein interactions during substrate catalysis.
Kirchhofer
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D
Departments of Cardiovascular Research and Protein Engineering, Genentech, Inc., South San Francisco, California 94080, USA. dak@gene.com
Lipari
M T
MT
Moran
P
P
Eigenbrot
C
C
Kelley
R F
RF
D000577:Amides / Q000378:metabolism; D000906:Antibodies / Q000276:immunology; D004789:Enzyme Activation; D005164:Factor IX / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013925:Thromboplastin / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000577:Amides; D000906:Antibodies; D010743:Phospholipids; D011994:Recombinant Proteins; D005164:Factor IX; D005170:Factor X; D013925:Thromboplastin