Factor IX: Difference between revisions
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In the presence of [[calcium ions the Gla domain]] (Calcium ions 5 and 6 and Gla residues 17 and 21) interacts with serine head group of phosphatidylserine located on the phospholipid membrane <ref>PMID:4528109</ref>. In the absence of metal ions the Gla domain is highly disordered and unstructured which indicated that the metal ions, stabilize the structure <ref>PMID:1538724</ref>. Three Mg2+ ions, (Mg-1, Mg-7, and Mg-8) are located on the surface of FIX and correspond to FX, three Ca2+ ions (Ca-1, Ca-7, and Ca-8). Each Mg2+ ion has a <scene name='Factor_IX/Gladomain_1/7'>bipyramidal coordination</scene> with a water molecule (Mg–O in FIX is 2.12 Å, whereas the Ca–O distances in FX is 2.41 Å) and a pair of Gla residues. | In the presence of [[calcium ions the Gla domain]] (Calcium ions 5 and 6 and Gla residues 17 and 21) interacts with serine head group of phosphatidylserine located on the phospholipid membrane <ref>PMID:4528109</ref>. In the absence of metal ions the Gla domain is highly disordered and unstructured which indicated that the metal ions, stabilize the structure <ref>PMID:1538724</ref>. Three Mg2+ ions, (Mg-1, Mg-7, and Mg-8) are located on the surface of FIX and correspond to FX, three Ca2+ ions (Ca-1, Ca-7, and Ca-8). Each Mg2+ ion has a <scene name='Factor_IX/Gladomain_1/7'>bipyramidal coordination</scene> with a water molecule (Mg–O in FIX is 2.12 Å, whereas the Ca–O distances in FX is 2.41 Å) and a pair of Gla residues. | ||
To identify the exact locations of bound Mg2+ ions, crystal structures comparisons were made between Mg2+-free and Mg2+-bound conditions. In the Mg2+-free conditions, Mg-1, Mg-7, and Mg-8, were replaced by Ca2+ ions which induced an elongation of the bond between the ion and an oxygen atom from a distance of 2.11 to 2.34 Å. This small change in distance induces a [[rotation of 4 degrees of FIX]]. This suggests that the | To identify the exact locations of bound Mg2+ ions, crystal structures comparisons were made between Mg2+-free and Mg2+-bound conditions. In the Mg2+-free conditions, Mg-1, Mg-7, and Mg-8, were replaced by Ca2+ ions which induced an elongation of the bond between the ion and an oxygen atom from a distance of 2.11 to 2.34 Å. This small change in distance induces a [[rotation of 4 degrees of FIX]]. This suggests that the magnesium ions induce a closed form conformation that contributes to the tight association of the Gla domain. This situation probably arises mainly from a difference in length between O–Mg–O and O–Ca–O bridges. | ||
The Gla domain perhaps also interacts with factor VIIIa via Mg2+-binding sites. Membrane bound FIXa forms an arched structure which is seen in the spatial relationship among the Gla, epidermal growth factor, and serine protease domains. This arched allows for the formation of a concave surface on the right side of FIXa and acts as a binding site for factor VIIIa. The Mg-8 ion points toward this concave surface making it ideal in the interaction between FIXa and GVIIIa. Similarly, [[Mg-8 may contribute to the binding to factors VIIa and X]]. | The Gla domain perhaps also interacts with factor VIIIa via Mg2+-binding sites. Membrane bound FIXa forms an arched structure which is seen in the spatial relationship among the Gla, epidermal growth factor, and serine protease domains. This arched allows for the formation of a concave surface on the right side of FIXa and acts as a binding site for factor VIIIa. The Mg-8 ion points toward this concave surface making it ideal in the interaction between FIXa and GVIIIa. Similarly, [[Mg-8 may contribute to the binding to factors VIIa and X]]. | ||
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{{STRUCTURE_1nl0| PDB=1nl0 | SCENE= }} | {{STRUCTURE_1nl0| PDB=1nl0 | SCENE= }} | ||
To further probe into the structure of FIX Gla domain, [[conformation-specific anti-Factor IX antibodies]] were utilized. Using an | To further probe into the structure of FIX Gla domain, [[conformation-specific anti-Factor IX antibodies]] were utilized. Using an | ||
<scene name='Factor_IX/Antifactor/1'>anti-Factor IX antibody</scene>, 10C12, which is then reformatted into a F(ab')2 form (two Fab fragments connected by a leucine zipper). This antibody is a calcium specific antibody for factor IX and provides information on the inhibition of membrane binding | <scene name='Factor_IX/Antifactor/1'>anti-Factor IX antibody</scene>, 10C12, which is then reformatted into a F(ab')2 form (two Fab fragments connected by a leucine zipper)<ref>PMID:14722079</ref>. This antibody is a calcium specific antibody for factor IX and provides information on the inhibition of membrane binding. The antibody interacts with the loop (Leu-6, Phe-9, and Val-10) of the ''γ''-carboxyglutamic acids residues bound to calcium ions in the Gla domain. | ||
The Gla domain of Factor IX consists of an N-terminal loop and three short ''ω''-<scene name='Factor_IX/Antifactor_2/2'>helixes</scene>(helix A: residues 14-19; helix B: residues 24-32; helix C: residues 35-45). The calcium ions are positioned between the loop and the A and B helices with calcium liganding providing the folding energy to align the loop properly. Helices A and B are connected by a tight turn and further stabilized by a conserved disulfide bond between residues 18 and 23 of the Gla domain. The two cysteine residues also form two hydrogen bonds (Tyr-45 to Cys-18, 3.10 Å; Tyr-45 to Cys-23, 2.88 Å) to the side chain of a conserved Tyr-45 of helix C, providing an anchor to bundle three helices together. This tyrosine residue and the disulfide bond are part of the hydrophobic core between helix C and helix A/B that includes Phe-25, Ala-28, Phe-41, and Trp-42, a cluster that provides further hydrophobic energy to bind helix C to helix A/B. All of these residues are conserved among Gla domains of vitamin K-dependent proteins. | The Gla domain of Factor IX consists of an N-terminal loop and three short ''ω''-<scene name='Factor_IX/Antifactor_2/2'>helixes</scene>(helix A: residues 14-19; helix B: residues 24-32; helix C: residues 35-45). The calcium ions are positioned between the loop and the A and B helices with calcium liganding providing the folding energy to align the loop properly. Helices A and B are connected by a tight turn and further stabilized by a conserved disulfide bond between residues 18 and 23 of the Gla domain. The two cysteine residues also form two hydrogen bonds (Tyr-45 to Cys-18, 3.10 Å; Tyr-45 to Cys-23, 2.88 Å) to the side chain of a conserved Tyr-45 of helix C, providing an anchor to bundle three helices together. This tyrosine residue and the disulfide bond are part of the hydrophobic core between helix C and helix A/B that includes Phe-25, Ala-28, Phe-41, and Trp-42, a cluster that provides further hydrophobic energy to bind helix C to helix A/B. All of these residues are conserved among Gla domains of vitamin K-dependent proteins. | ||
The binding of FIX to the phospholipid membrane is carried out by the loop, in the N terminus of the Gla domain. Four Ca2+ ions from Ca-2 to Ca-5 are essential to stabilize the -loop region for membrane-binding | The binding of FIX to the phospholipid membrane is carried out by the loop, in the N terminus of the Gla domain. Four Ca2+ ions from Ca-2 to Ca-5 are essential to stabilize the ''ω''-loop region for membrane-binding <ref>PMID:8069632</ref> because these Ca2+ ions bind to conserved Gla7 and Gla8 residues in this region. The replacement by Mg2+ ions may lead to destabilization of the structure of the Gla domain, particularly in this region because of significant differences that exist with Ca2+ ion in both coordination distance and coordination sphere (Mg2+ ion takes generally a typical bipyramidal configuration and six coordination). This may explain why the Ca2+ ions are not replaced by Mg2+ ions in the present structure. | ||
The interaction of the Gla domain with the phospholipid membrane is carried out by binding to phosphatidylserine in the membrane through the interaction of calcium ions. Upon calcium binding to FIX a conformational change occurs | The interaction of the Gla domain with the phospholipid membrane is carried out by binding to phosphatidylserine in the membrane through the interaction of calcium ions. Upon calcium binding to FIX a conformational change occurs in the Gla domain through the clustering of N-terminal hydrophobic residues into a hydrophobic patch, which is then exposed to the solvent. This hydrophobic patch then allows the association of the Gla domain with the cell surface membrane through electrostatic interactions between the phosphoserine head group and arginine and lysine residues in the Gla domain. The basic amino acid residues of factor IX (Lys-5 and Arg-10) bind to the glycerol phosphate backbone and the carboxyl group of the serine interacts with Ca-5 and Ca-6<ref>PMID:500729</ref> allowing membrane association. | ||
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== Serine Protease Domain == | == Serine Protease Domain == | ||
{{STRUCTURE_1rfn| PDB=1rfn | SCENE= }} | {{STRUCTURE_1rfn| PDB=1rfn | SCENE= }} | ||
The serine protease domain of FIX is required for blood coagulation. FIX circulates in plasma as a single-chain zymogen at a concentration of 2.5-5 mg/ml and a half life is approximately 24 hours. The zymogen is activated by either FVII–tissue-factor complex or Factor XIa (FXIa). During activation the peptide bonds | The serine protease domain of FIX is required for blood coagulation<ref>PMID:10467178</ref>. FIX circulates in plasma as a single-chain zymogen at a concentration of 2.5-5 mg/ml and a half life is approximately 24 hours. The zymogen is activated by either FVII–tissue-factor complex or Factor XIa (FXIa). During activation the peptide bonds between Arg145–Ala146 and Arg180–Val181 are cleaved releasing an 11 kDa activation peptide from the Factor IX. This cleavage allows the exposure of the serine protease site on the heavy chain which can then activate Factor X in the presence of Factor VIII, calcium and phospholipid surface. This enzyme belongs to the family of trypsin-like serine proteases.The mechanism of these serine proteases involves the catalytic triad, which is found in the enzymes active site and is composed of three amino acids. | ||
The serine protease domain of FIX accounts for half of its mass and contains a conserved catalytic triad made of Asp, His, and Ser. The binding pockets of these vitamin-K depended proteases recognize a small number of amino acids sequences allowing them to cleave at arginyl residues with high substrate specificity. Serine’s hydroxyl group carries out the nucleophillic attack. While the imidazol ring of hisidine takes up the liberated proton and the carboxylate ion of Asp stabilizes the developing charge. Unlike other serine protease family members these vitamin K dependent proteases have an extended specificity pocket which allows a small number of amino acids to be recognized. | The serine protease domain of FIX accounts for half of its mass and contains a conserved catalytic triad made of Asp, His, and Ser. The binding pockets of these vitamin-K depended proteases recognize a small number of amino acids sequences allowing them to cleave at arginyl residues with high substrate specificity. Serine’s hydroxyl group carries out the nucleophillic attack. While the imidazol ring of hisidine takes up the liberated proton and the carboxylate ion of Asp stabilizes the developing charge. Unlike other serine protease family members these vitamin K dependent proteases have an extended specificity pocket which allows a small number of amino acids to be recognized. | ||