Crystal Structure of Mg2+- and Ca2+-bound Gla Domain of Factor IX Complexed with Binding Protein

Nitrophorin 2 (NP2) is a salivary lipocalin from Rhodnius prolixus that binds with coagulation factors IX (fIX) and IXa (fIXa). Binding of NP2 with fIXa results in potent inhibition of the intrinsic factor Xase complex. A panel of site-directed surface mutants of NP2 was generated to locate determinants of high affinity fIX(a) binding. The locations of the mutations were based on comparisons with the related, but less potent, inhibitor nitrophorin 3 (NP3). Three point mutants (K21A, K92A, and V94A) were found that clearly reduced the inhibitory potency as measured by the activity of a reconstituted factor Xase system. Binding of NP2 with fIXa and fIX as measured by surface plasmon resonance and isothermal titration calorimetry was reduced in a similar manner. Of the three mutants, two (K92A and V94A) were located on the loop connecting ␤-strands E and F of the lipocalin ␤-barrel. The largest changes were seen with the K92A mutation, which lies at the apex of the loop, with a smaller effect being seen with mutation of Val 94 . Combination of four E-F loop mutations (K92A, A93K, V94A, E97A) in a single mutant reduced the inhibitory potency and binding to levels similar to those seen with NP3 without affecting heme or histamine binding. The intrinsic factor Xase complex (fXase)1 is an essential component of the mammalian blood coagulation cascade consisting of factors IXa (fIXa), VIIIa (fVIIIa), and X (fX) assembled on an anionic phospholipid surface (1). Disruption of this reaction results in two of the most important human bleeding disorders, hemophilias A (fVIII deficiency) and B (fIX deficiency) (1). Nitrophorin 2 (NP2) is a salivary anticoagulant protein from the blood-feeding insect Rhodnius prolixus that targets the intrinsic fXase complex (2, 3). It is a hyperbolic mixed-type inhibitor that acts by interfering with assembly of the proteolytic complex and/or phospholipid binding (4). NP2 does not inhibit the amidolytic activity of fIXa but rather blocks fXa formation in reactions lacking either phospholipid vesicles or fVIIIa (4). Factor X activation is not inhibited in the absence of fVIIIa and phospholipid suggesting that interactions between fX and fIXa are not directly influenced by NP2, while those between fIXa and fVIIIa/phospholipid are (4). Nevertheless, direct binding of NP2 with fIX and fIXa has been shown using surface plasmon resonance (SPR), while in the same study, no interaction of NP2 with fVIII was seen (5). Binding with fIX also results in inhibition of fIXa generation by both fXIa and the tissue factor-fVIIa complex (5). Removal of the Gla (␥-carboxyglutamic acid-rich) domain of fIX was found to eliminate the SPR interaction, suggesting that the NP2 binding site is located at or near this region, a primary point of interaction between fIXa and the phospholipid surface (5) and fVIIIa (6).NP2 is a member of the nitrophorin (NP) group, consisting of four related salivary lipocalins in R. prolixus that are remarkable in taking on numerous functions related to blood feeding (7-10). NPs are heme...

Section: Resultsmentioning

confidence: 69%

Structural Determinants of Factor IX(a) Binding in Nitrophorin 2, a Lipocalin Inhibitor of the Intrinsic Coagulation Pathway

Gudderra

Ribeiro

Andersen

2005

“…Recently, the structure of bovine Factor IX-(1-46) bound to a snake venom protein, Factor IX-binding protein, has been crystallized in the presence of calcium and magnesium ions and in the presence of calcium alone (9). Although the emphasis of this work is on the possible role of magnesium in Gla domain binding to membrane surfaces, examination of the calciumFactor IX- complex revealed that the calcium coordination within the Gla domain differs from the Gla domains of other vitamin K-dependent proteins, including prothrombin (7), Factor VII (10), and Factor X (11).…”

mentioning

confidence: 99%

Crystal Structure of the Calcium-stabilized Human Factor IX Gla Domain Bound to a Conformation-specific Anti-factor IX Antibody

Huang

Furie

2004

“…7B. These data indicate that the minimum epitope recognized by B1 cannot be formed by phosphoserine alone, but also requires hydrophobic amino acids located at the amino and carboxyl ends of peptide AP- (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). It is likely that the three-dimensional structure of this epitope is formed by hydrophobic interactions of these residues.…”

Section: Discussionmentioning

confidence: 89%

“…The AP-containing fractions were applied to an IX-bp-Cellulofine column in the presence of 1 mM Ca 2ϩ to remove intact FIX, FIXa␣, and FIXa␤. Molecules that maintain the Gla domain bound to IX-bp-Cellulofine in the presence of Ca 2ϩ ions (15). Breakthrough fractions containing pAP were then applied to a reverse phase HPLC column.…”

Section: Resultsmentioning

confidence: 99%

Characterization of a Monoclonal Antibody B1 That Recognizes Phosphorylated Ser-158 in the Activation Peptide Region of Human Coagulation Factor IX

Atoda

Yokota

Morita

2006

Self Cite

Blood coagulation factor IX (FIX) undergoes various post-translational modifications such as ␥-carboxylation and glycosylation. Non-phosphorylated recombinant FIX has been reported to rapidly disappear from plasma, indicating that phosphorylation of FIX plays an important role in the physiological activity of this coagulation factor. In this study, we characterized the human FIX activation peptide (AP) using a monoclonal antibody that recognizes phosphorylated Ser-158 in the AP region. Murine monoclonal antibody B1 against human FIX recognized FIX with an apparent K d value of 5 nM in the presence of Ca 2؉ (EC 50 ‫؍‬ 0.58 mM). B1 bound to the isolated AP of FIX and retained the Ca 2؉ dependence of binding to the isolated AP. The deglycosylation of AP did not affect the binding of B1 to AP, while B1 failed to bind to recombinant AP expressed in Escherichia coli. MALDI-TOF mass spectrometry showed that the m/z of plasma-derived deglycosylated AP is 82.54 Da greater than that of recombinant AP. The binding ability of B1 to AP was lost by the dephosphorylation of plasma-derived AP. B1 bound to synthetic peptide AP-(5-19), including phosphoserine-13, but not to the non-phosphorylated AP-(5-19) in the presence of Ca 2؉ . These data provide direct evidence that Ser-13 of the plasma-derived FIX AP region (Ser-158 of FIX) is phosphorylated and that B1 recognizes the epitope, which includes Ca 2؉ -bound phosphoserine-158. B1 should be useful in the quality control of biologically active recombinant FIX containing phosphoserine-158. Blood coagulation factor IX (FIX)2 is present in an inactive precursor form that consists of a ␥-carboxyglutamic acid (Gla)-containing domain, two epidermal growth factor (EGF)-like domains, an activation peptide (AP) region, and a serine protease domain (1) (Fig. 1). FIX is activated physiologically by factor XIa or factor VIIa-tissue factor complex to generate FIXa␤ via FIX␣, an inactive intermediate, whereas RVV-X, a protease from Russell's viper venom, can activate FIX to FIXa␤ via FIXa␣, an active intermediate (2). In both cases, AP is removed by peptide bond cleavage at two sites of FIX to generate the final form of activated FIX, FIXa␤, and an AP. Increased plasma FIX level is one of the risk factors of thrombosis (3), and along with the concentration of AP in plasma is a useful index for the activation of the blood coagulation system.Human coagulation FIX undergoes various post-translational modifications including ␥-carboxylation of twelve Glu residues in the Gla domain, attachment of N-linked and O-linked oligosaccharides at the EGF domain and AP region, and ␤-hydroxylation of an Asp residue in the first EGF domain (4). Other post-translational modifications, such as sulfation of a Tyr residue and phosphorylation of Ser residue(s), also have been suggested (5) but precise data on these have not been reported to date. These post-translational modifications take place in the liver and may play important roles for specific activity, secretion, recovery, half-life in the circulation, and ...