Activation and active site occupation alter conformation in the region of the first epidermal growth factor-like domain of human factor VII.
The first epidermal growth factor-like domain (EGF-1) of factor VII (FVII) provides the region of greatest contact during the interaction of FVIIa with tissue factor. To understand this interaction better, the conformation-sensitive FVII EGF-1-specific monoclonal antibody (mAb) 231-7 was used to investigate the conformational effects occurring in this region upon both FVII activation and active site occupation. The binding affinity of mAb 231-7 was approximately 3-fold greater for the zymogen state than for the active state; a result affected by the presence of both calcium and the adjacent Gla domain. Once activated, active site inhibition of FVIIa with a variety of chloromethyl ketone inhibitors resulted in a 10-fold range of affinities of FVIIai molecules to mAb 231-7. Gla domain removal eliminated this variation in affinity, suggesting the involvement of a Gla/EGF-1 interaction in this conformational effect. In addition, the binding of mAb 231-7 to FVIIa EGF-1 stimulated the amidolytic activity of free FVIIa. Taken together, these results imply an allosteric interaction between the FVIIa active site and the EGF-1 domain that is sensitive to variation in active site occupant structure. Thus, these present studies indicate that the conformational change associated with FVII activation and active site occupation involves the EGF-1 domain and suggest potential functional consequences of these changes.
Leonard
B J
BJ
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario L8S 3Z5, Canada.
Clarke
B J
BJ
Sridhara
S
S
Kelley
R
R
Ofosu
F A
FA
Blajchman
M A
MA
D000495:Allosteric Site; D000590:Amino Acid Chloromethyl Ketones / Q000037:antagonists & inhibitors; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000737:chemistry; D005167:Factor VII / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013925:Thromboplastin / Q000737:chemistry / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D005167:Factor VII; D013925:Thromboplastin; D002118:Calcium