Ca2+ binding to the first epidermal growth factor module of coagulation factor VIIa is important for cofactor interaction and proteolytic function.
Epidermal growth factor-like (EGF) domain Ca2+ binding sites in the homologous coagulation factors VII, IX, and X stabilize the structural orientation of the gamma-carboxyglutamic acid-rich (Gla) domain relative to EGF-1. Site-directed mutagenesis was employed here to analyze the functional importance of Ca2+ binding to EGF-1 in factor VIIa (VIIa), which initiates coagulation in complex with its cofactor, tissue factor (TF). Ala replacements for Asp63 or Gln49 resulted in reduced TF affinity concordant with the number of eliminated Ca2+-coordinating oxygen atoms in the respective side chains. Ca2+ binding to EGF-1 had no major direct effect on contacts with TF residue Gln110 or on interactions of VIIa residues Arg79 and Phe40, suggesting that the stabilized Gla-EGF-1 orientation affects overall docking. Gly, Ala, and Glu replacements at Asp46, which is a Ca2+-coordinating residue at the Gla aromatic stack carboxyl terminus, are consistent with the notion that an increased flexibility of the Gla domain relative to EGF-1 contributes significantly to loss of function. Certain mutants in the EGF-1 Ca2+ site had reduced proteolytic function, suggesting the importance of the high affinity Ca2+ binding site for macromolecular substrate interaction.
Kelly
C R
CR
Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Dickinson
C D
CD
Ruf
W
W
D015055:1-Carboxyglutamic Acid / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005164:Factor IX / Q000378:metabolism; D015942:Factor VIIa / Q000737:chemistry / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D005973:Glutamine; D015055:1-Carboxyglutamic Acid; D004815:Epidermal Growth Factor; D001216:Asparagine; D005164:Factor IX; D005170:Factor X; D015942:Factor VIIa; D000409:Alanine; D002118:Calcium; D005998:Glycine