The integrity of the cysteine 186-cysteine 209 bond of the second disulfide loop of tissue factor is required for binding of factor VII.
The structural basis of function of tissue factor (TF), the cell surface receptor and cofactor for the serine protease factor VIIa, cannot be inferred from the primary sequence. The functional significance of the two disulfide bonded loops in the surface domain of TF has been analyzed using site-directed mutagenesis to selectively preclude covalent stabilization of these loops by pairwise substitution of serine residues for cysteines. Mutant TF lacking either the amino (TFS49S57) or carboxyl (TFS186S209) disulfide bond were expressed on the surface of cells consistent with proper processing. Each reacted with a panel of monoclonal antibodies further suggesting proper global folding of the mutant proteins. TFS186S209 exhibited a selective decrease in reactivity with an antibody directed against one epitope locus in the carboxyl aspect of the surface domain of TF. Whereas TFS49S57 was functionally comparable to the wild type protein, TFS186S209 was functionally 30-40-fold less effective, and the affinity of factor VIIa binding to this mutant was indirectly estimated to be diminished 20-fold. These data suggest that the Cys186-Cys209 disulfide bond is required to maintain conformation and implicate the disulfide loop or adjacent structures in the carboxyl half of the surface domain of TF in receptor function.
Rehemtulla
A
A
Vascular Cell and Molecular Biology Program, Department of Immunology, La Jolla, California 92037.
Ruf
W
W
Edgington
T S
TS
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D005167:Factor VII / Q000378:metabolism*; D005434:Flow Cytometry; D008969:Molecular Sequence Data; D009154:Mutation; D013925:Thromboplastin / Q000737:chemistry* / Q000235:genetics
D004220:Disulfides; D005167:Factor VII; D013925:Thromboplastin; D003545:Cysteine