Identification of the Phospholipid Binding Site in the Vitamin K-dependent Blood Coagulation Protein Factor IX

Abstract

The blood coagulation and regulatory proteins that contain ␥-carboxyglutamic acid are a part of a unique class of membrane binding proteins that require calcium for their interaction with cell membranes. Following protein biosynthesis, glutamic acids on these proteins are converted to ␥-carboxyglutamic acid (Gla) in a reaction that requires vitamin K as a cofactor. The vitamin K-dependent proteins undergo a conformational transition upon metal ion binding, but only calcium ions mediate protein-phospholipid interaction. To identify the site on Factor IX that is required for phospholipid binding, we have determined the three-dimensional structure of the Factor IX Gla domain bound to magnesium ions by NMR spectroscopy. By comparison of this structure to that of the Gla domain bound to calcium ions, we localize the membrane binding site to a highly ordered structure including residues 1-11 of the Gla domain. In the presence of Ca 2؉ , Factor IX Gla domain peptides that contain the photoactivatable amino acid p-benzoyl-L-phenylalanine at positions 6 or 9 cross-link to phospholipid following irradiation, while peptides lacking this amino acid analog or with this analog at position 46 did not cross-link. These results indicate that the NH 2 terminus of the Gla domain, specifically including leucine 6 and phenylalanine 9 in the hydrophobic patch, is the contact surface on Factor IX that interacts with the phospholipid bilayer.

Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex

“…FVIIIa binds to phospholipid via the C2 domain 33,34 and FIXa binds to the phospholipid surface via the amino-terminal 3-11 residues of the Gla domain. 35,36 This association facilitates the orientation and interaction of the proteins and has the effect of increasing the affinity of the FVIIIa-FIXa interaction. [37][38][39] The FVIIIa-FIXa interaction in the absence of phospholipid is less efficient, with the rate enhancement component due to the orientation effect of phospholipid removed.…”

Molecular Diversity in Venom from the Australian Brown Snake, Pseudonaja textilis

“…This modification is conserved across different species (34,32). The Gla residues chelate calcium ions and induce a conformational transition in the Gla domain that results in phospholipid membrane binding and stimulation of the procoagulant activity of the prothrombinase complex (35). As well as mammalian coagulation factors, Gla domains have been found in the toxins of predatory cone snails of the genus Conus (36,37).…”

Mutagenesis of the γ-Carboxyglutamic Acid Domain of Human Factor VII to Generate Maximum Enhancement of the Membrane Contact Site

“…The results indicate that nearly all functional improvements arose from membrane binding affinity. The largest enhancements in membrane affinity resulted from mutation in the region of residue 32, a position that is located quite far from the N-terminal end of the protein, where membrane contact is generally suggested (39). The mutant with highest function, (Y4)P10Q/K32E/D33F/A34E, showed 150 -296-fold improvement over wild-type FVII.…”