Mutagenesis of the gamma-carboxyglutamic acid domain of human factor VII to generate maximum enhancement of the membrane contact site.
Site-directed mutagenesis of the 40 N-terminal residues (gamma-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 gamma-carboxyglutamic acid at these positions. Except for the Tyr(4) 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.
Harvey
Stephen B
SB
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis 55455, USA.
Stone
Matthew D
MD
Martinez
Michael B
MB
Nelsestuen
Gary L
GL
D015055:1-Carboxyglutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D005167:Factor VII / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015055:1-Carboxyglutamic Acid; D005167:Factor VII; D005170:Factor X