Ca2+ binding to the first epidermal growth factor module of coagulation factor VIIa is important for cofactor interaction and proteolytic function.

Curtis R Kelly,Craig D Dickinson,Wolfram Ruf

doi:10.1074/jbc.272.28.17467

Curtis R Kelly, Craig D Dickinson + Show 1 more

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https://doi.org/10.1074/jbc.272.28.17467

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Abstract

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.

Highlights

EGF1 modules function in Ca2ϩ-dependent extracellular protein-protein interactions mediating cell adhesion and activation of protease cascades
The expression levels typically achieved in transient transfection experiments were below concentrations needed to achieve full saturation of tissue factor (TF) by the Asp63 3 Ala mutant, indicating a severe reduction in TF binding and possibly diminished proteolytic function (Table I)
Whereas Ca2ϩ has little impact on the conformation of the TF extracellular domain [17], Ca2ϩ binding to the Gla domain is important for high affinity binding to TF

Summary

Introduction

EGF1 modules function in Ca2ϩ-dependent extracellular protein-protein interactions mediating cell adhesion and activation of protease cascades. Whereas fluorescence quenching indicated a ϳ30 ␮M affinity for the EGF-1 site, terbium phosphorescence measurements suggested a Ca2ϩ affinity of ϳ2 mM for the catalytic domain site [11, 13] This higher estimate for the catalytic domain site likely results from the experimental conditions and is inconsistent with the Ca2ϩ-dependent changes in the amidolytic function of VIIa which are attributable to saturation of the protease domain Ca2ϩ site with a midpoint of ϳ50 –250 ␮M Ca2ϩ [11, 14, 15]. We demonstrate that Ca2ϩ binding to this site is responsible for an increased affinity of VIIa for TF and provide evidence in support of the hypothesis that Ca2ϩ coordination in EGF-1 may play a functional role through the stabilization of the orientation of the Gla domain relative to EGF-1

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