Mutagenesis of the γ-carboxyglutamic acid domain of human factor VII to generate maximum enhancement of the membrane contact site

Stephen B Harvey, Matthew D. Stone, Michael B. Martinez, Gary L Nelsestuen

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Site-directed mutagenesis of the 40 N-terminal residues (γ-carboxyglutamic acid domain) of blood clotting factor VII was carried out to identify sites that improve membrane affinity. Improvements and degree of change included P10Q (2-fold), K32E (13-fold), and insertion of Tyr at position 4 (2-fold). Two other beneficial changes, D33F (2-fold) and A34E (1.5-fold), may exert their impact via influence of K32E. The modification D33E (5.2-fold) also resulted in substantial improvement. The combined mutant with highest affinity, (Y4)P10Q/K32E/D33F/ A34E, showed 150-296-fold enhancement over wild-type factor VIIa, depending on the assay used. Undercarboxylation of Glu residues at positions 33 and 34 may result in an underestimate of the true contributions of γ-carboxyglutamic acid at these positions. Except for the Tyr4 mutant, all other beneficial mutations were located on the same surface of the protein, suggesting a possible membrane contact region. An initial screening assay was developed that provided faithful evaluation of mutants in crude mixtures. Overall, the results suggest features of membrane binding by vitamin K-dependent proteins and provide reagents that may prove useful for research and therapy.

Original languageEnglish (US)
Pages (from-to)8363-8369
Number of pages7
JournalJournal of Biological Chemistry
Volume278
Issue number10
DOIs
StatePublished - Mar 7 2003

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Mutagenesis
Factor VII
Human engineering
Membranes
Acids
Assays
Factor VIIa
Blood Coagulation Factors
Vitamin K
Blood Coagulation
Site-Directed Mutagenesis
Screening
Membrane Proteins
Blood
Mutation
Research
Proteins
Therapeutics

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Mutagenesis of the γ-carboxyglutamic acid domain of human factor VII to generate maximum enhancement of the membrane contact site. / Harvey, Stephen B; Stone, Matthew D.; Martinez, Michael B.; Nelsestuen, Gary L.

In: Journal of Biological Chemistry, Vol. 278, No. 10, 07.03.2003, p. 8363-8369.

Research output: Contribution to journalArticle

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