Characterization of γ-Carboxyglutamic Acid Residue 21 of Human Factor IX

Abstract

We investigated the functional role of gamma-carboxyglutamic acid (Gla) residue 21 of human factor IX, using site-directed mutagenesis to change the glutamic acid residue to aspartic acid (FIX21D). FIX21D had reduced activity in an activated partial thromboplastin time (aPTT) assay and was activated by factor XIa more slowly than wild-type factor IX (FIXwt). FIX21D underwent normal, two-stage calcium-dependent intrinsic fluorescence quenching, indicating that a folding event similar to that seen in FIXwt occurred upon the addition of calcium ions. Antibody A-7, which recognizes factor IX-specific residues at positions 33-40, bound FIX21D as well as FIXwt; however, the calcium-specific monoclonal antibody, JK-IX-2, whose epitope includes residues 1 and 22, did not recognize FIX21D. FIX21D bound phosphatidylserine/phosphatidylcholine (PS/PC) vesicles with Kd approximately 10-fold greater than FIXwt, as measured by a fluorescence light scattering assay. Finally, although FIXwt binds endothelial cells with a Kd of 2.8 nM, FIX21D did not bind endothelial cells. Molecular modeling simulations of FIXwt and FIX21D indicate that mutating Gla 21 to Asp causes structural changes in residues 3-5 and 8-10, as well as in two exposed calcium ions, consistent with the reduced function of FIX21D. Immunological and intrinsic fluorescence quenching assays and the molecular dynamics simulations suggest normal folding in the C-terminal region of the Gla domain. Thus we hypothesize the FIX21D has reduced JK-IX-2 and phospholipid and endothelial cell binding due to localized structural changes in residues 3-10 and the exposed calcium ions. Our study suggests that the Gla 21 to Asp mutation disrupts function in the N-terminal region of the Gla domain without affecting structure in the C-terminal Gla domain region.

X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B.

“…Several lines of evidence (20,21,(44)(45)(46)(47)(48) suggest that in the absence of calcium the interaction between EGF-1 and the C-terminal helix of the Gla domain (i.e., the hydrophobic stack) is weak; the helical topology of the Gla domain conforms to the calcium-bound Gla domain on average, albeit with higher mobility of the secondary structure elements (25,26). The crystal structure of prothrombin fragment 1 determined in the absence of calcium placed the a-helical hydrophobic stack adjacent to the kringle 1 domain (20); calcium binding induces a conformational transition to a more ordered structure, with several Gla residues becoming partially buried and membrane binding determinants such as Leu-6 or Phe-9 exposed (21,25,26).…”

“…As in prothrombin (21), the first 9 Gla residues together with the exposed hydrophobic residues of the fQ-loop (Fig. la) are most likely responsible for calcium-dependent phospholipid membrane binding in a not fully understood manner (21,25,26,(44)(45)(46)(47)(48). Another important aspect is the interaction of fIXa with cofactor Vllla and substrate fX.…”

“…The N-terminal light chain (145 residues) and the C-terminal heavy chain (235 residues) are disulfide linked via Cys-132-Cys-289(c122). The light chain consists of several modules, which also reflect the exon structure (9): the N-terminal Gla module (residues 1-38; Gla refers to Cy carboxylated glutamic acid residues) followed by its hydrophobic helix (39)(40)(41)(42)(43)(44)(45)(46), two epidermal growth factor (EGF)-like domains (residues 47-84 and 85-127), and a linker peptide (residues 128-145); the heavy chain (residues 181-415) forms the catalytic module. Free fIXaf3 is an extremely poor proteinase (10-14) but becomes a potent fX activator upon complex formation with its cofactor fVIIIa via calciummediated binding in vitro to anionic phospholipid membranes and in vivo to the surfaces of endothelial cells or activated platelets (15).…”

Calcium Binding to a Factor IX Fc Fusion Protein and Effects on Higher‐Order Structure

“…The binding of Ca 2+ ions is required for physiologic activation of factor IX and abnormalities in the calcium‐binding region have been shown to decrease clotting activity 15. A wide range of methodologies have been applied to characterize Ca 2+ binding to factor IX: amino acid substitution studies have probed the functional significance of specific Ca 2+ ‐binding residues,16 X‐ray crystal structures including calcium‐bound forms have been reported for factor IX,17–19 as well as H 1 NMR studies of the Gla domain20 and first EGF‐like domain,21 which have provided three‐dimensional solution structures of specific Ca 2+ ‐binding sites. The Ca 2+ ‐binding stoichiometries of the intact factor IX molecule22 and individual domains23–25 have been reported, although to our knowledge characterization of Ca 2+ binding by isothermal titration calorimetry (ITC) has not been reported.…”

Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography

“…Conformational changes have been also observed in all the VKD coagulation proteins using fluorescence quenching due to specific X 2+ site binding [8,16,17]. FIX is an example of a VKD protein with X 2+ binding sites not only in the Gla domain but it also contains two additional Gla residues that help to form an additional Mg 2+ site [18-20]. FIX also contains a Ca 2+ site in one of two epidermal growth factor-like domains and also in its C-terminal serine protease domain [1,2].…”