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Factor VII mutant V154G models a zymogen-like form of factor VIIa
Raffaella Toso1, Francesco Bernardi, Theresa Tidd
1Department of Biochemistry and Molecular Biology, University of Ferrara, Via Luigi Borsari, 46 Ferrara 44100, Italy. r-toso@hotmail.com
Abstract:
Proteolytic cleavage of the peptide bond between Arg(152) and Ile(153) converts the procoagulant protein Factor VII (FVII) to an activated two-chain form (FVIIa). The formation of a salt bridge between Ile(153) and Asp(343) drives the conversion of FVIIa from being zymogen-like to the active form. In the present paper, we describe the novel FVII mutant V154G (Val(154)-->Gly mutation; residue 17 in the chymotrypsin numbering system), found in three FVII-deficient patients, which models a zymogen-like form of FVIIa. Recombinant V154G FVIIa, although normally cleaved, shows markedly reduced activity towards peptidyl substrate and undetectable activity towards macromolecular substrates. Susceptibility of Ile(153) to chemical modification, in either the presence or the absence of tissue factor (TF), suggests that the reduced V154G FVIIa activity is caused by impaired salt-bridge formation, thus resulting in a zymogen-like FVIIa form. The TF-mediated protection from chemical modification of V154A indicated that Gly(154) is responsible for this peculiar feature, and suggests that this region, proximal to the heavy chain N-terminus, is directly involved in the conversion of FVII into FVIIa. V154G FVII was exploited to study the FVII-TF interaction, together with three additional FVII variants that were expressed to serve as models for different FVII forms. The comparison of binding affinities of full-length TF after relipidation in L-alpha-phosphatidylcholine for the zymogen FVII (Arg(152)-->Gln, K (d)=1.04+/-0.27 nM), inactive FVIIa (Ser(344)-->Ala, K (d)=0.27+/-0.06 nM) and a zymogen-like FVIIa (V154G, K (d)=1.15+/-0.16 nM) supports the hypothesis that preferential binding of TF to active FVIIa is insufficient to drive the 10(5)-fold enhancement of FVIIa activity. In addition, the inability of V154G FVIIa to accommodate an inhibitor in the active site, indicating an improperly shaped specificity pocket, would explain the low activity of the zymogen-like form of FVIIa, which is predominant in the absence of TF.
Insights
A novel Factor VII (FVII) mutation, V154G, creates a zymogen-like FVIIa form with significantly reduced activity. This finding highlights the importance of specific salt bridges in FVIIa activation and function.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- Factor VII (FVII) is a procoagulant protein activated by proteolytic cleavage.
- Activation converts FVII to FVIIa, involving a salt bridge formation crucial for its active conformation.
- Understanding FVIIa activation mechanisms is vital for hemostasis research.
Purpose of the Study:
- To characterize a novel FVII mutant, V154G, found in FVII-deficient patients.
- To investigate the role of residue 154 in FVIIa activation and function.
- To explore the FVII-TF interaction and its impact on FVIIa activity.
Main Methods:
- Site-directed mutagenesis to create the V154G FVII mutant.
- Enzymatic activity assays using peptidyl and macromolecular substrates.
- Chemical modification susceptibility studies with and without tissue factor (TF).
- Binding affinity studies of FVII variants with TF.
Main Results:
- The V154G FVIIa mutant exhibits markedly reduced peptidyl substrate activity and undetectable macromolecular substrate activity.
- Impaired salt-bridge formation between Ile(153) and Asp(343) is suggested by chemical modification data.
- V154G FVIIa shows a zymogen-like form with altered TF interaction and an improperly shaped active site pocket.
- Binding affinity studies reveal TF binding is insufficient to drive the full enhancement of FVIIa activity.
Conclusions:
- The V154G mutation results in a zymogen-like FVIIa with impaired catalytic activity due to disrupted salt bridge formation.
- Residue 154 plays a critical role in FVIIa activation and TF-mediated protection.
- The findings provide insights into the structural requirements for FVIIa activation and function.