Factor IX: Difference between revisions

<span style="color:Brown">'''<scene name='Factor_IX/Ixstructure/3'>Factor IX</scene>'''</span> (plasma thromboplastin component, Christmas factor, or hemophilia B factor) is a 57-kDa vitamin K-dependent procoagulant glycoprotein. It is synthesized by the liver hepatocyte as a [[pre-prozymogen]] that requires extensive posttranslational modification<ref>PMID:2169923</ref>. The [[pre-prozymogen]] contains a pre-peptide (hydrophobic signal peptide) at its amino terminal that transports the growing polypeptide into the lumen of the Endoplasmic Reticulum. Once inside the ER, this signal peptide is cleaved by a signal peptidase. 

<scene name='Factor_IX/Ixstructure_residue/2'>Gla</scene>) residues<ref>PMID:12554099</ref>. These residues are required for the association with the anionic phospholipid surface through Ca2+-dependent binding.  

<scene name='Factor_IX/Ixstructure_egf1/2'>EGF-1</scene> and <scene name='Factor_IX/Ixstructure_egf2/2'>EGF-2</scene>). The N-terminus of <scene name='Factor_IX/Ixstructure_egf1/2'>EGF-1</scene> contains a Ca2+ binding site, while the C-terminus connects to a hydrophobic pocket of <scene name='Factor_IX/Ixstructure_egf2/2'>EGF-2</scene> by a salt bridge through Lys122 (<scene name='Factor_IX/Ixstructure_egf1/2'>EGF-1</scene> residue) and Gln74 (<scene name='Factor_IX/Ixstructure_egf2/2'>EGF-2</scene>)<ref>PMID:11723140</ref>. <scene name='Factor_IX/Ixstructure_egf2/2'>EGF-2</scene> connects to the <scene name='Factor_IX/Ixstructure_chainc/2'>serine protease</scene> domain through a linker peptide that is required for a proper orientation and folding of <scene name='Factor_IX/Ixstructure_chainc/2'>serine proteases</scene>. To have a physiologically active factor IX, two cleavages must occur to remove a 35 amino acid region that precedes the catalytic site.  

The first cleavage is at Arg145, generating an inactive FIXα. The second cleavage is at Arg180 results in a catalytically active molecule FIXaβ. This resulting heterodimer is held by a disulfide bridge at Cys132-Cys289. The <scene name='Factor_IX/Ixstructure_chainc/2'>serine protease</scene> contains a catalytic triad of <scene name='Factor_IX/Ixstructure_catalytictriad/2'>His221, Asp269, and Ser365</scene><ref>PMID:9374470</ref>. Upon cleave at Arg180, Val181 can form a salt bridge with Asp364, which is a characteristic of active <scene name='Factor_IX/Ixstructure_chainc/2'>serine proteases</scene>. The active FIXa, can then interact with its cofactor, FVIIIa, to form a membrane-bound Xase complex, which activated FX to FXa.

Exon I encodes the hydrophobic signal peptide that targets the FIX into the lumen of the Endoplasmic Reticulum. Exon II codes for the pro-peptide and the Gla domain. Exon III encodes the <scene name='Factor_IX/Ixstructure_residue39_46/3'>hydrophobic helix</scene>, that inserts itself into the lipid membrane, anchoring FIX. Exon IV and V, code for EGF-1 and EGF-2, the serine protease region is encoded by exon VI-VIII.

 

{{STRUCTURE_1j35| PDB=1j35  |  SCENE=  }}

The Gla domain is situated at the N-terminus of coagulation factor IX, and is evolutionary conserved in other vitamin K dependent proteins such as factor VII, X, and prothrombin. A <scene name='Factor_IX/Gladomain_glamgca/2'>Gla domain</scene> is made up of 10-13 of γ-carboxyglutamic acid residues and requires both <scene name='Factor_IX/Gladomain_1/6'>Ca2+ and Mg2+ </scene> ions for membrane association and stabilization of its active three dimensional conformation <ref>PMID:3511981</ref>.  

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 magnisuim 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.  

Bovine <scene name='Factor_IX/Gladomain/1'>Factor IX -(1-46)</scene> bound to a snake venom protein (Factor IX-bp), is crystallized in the presence of calcium and magnesium ions and in the presence of calcium alone.  

<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.  by this conformation-specific antibody. The antibody interacts with the  loop (Leu-6, Phe-9, and Val-10)  of the  -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 ''&omega;''-<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 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 I 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 (2) allowing membrane association.

 

 

 

 

 

The catalytic domain of FIX is composed of two β-<scene name='Factor_IX/Rfnscene/3'>barrel</scene> subdomains that form an active site at their interface. The active site is located at the junction of these β-barrels. The EGF-2 domain is connected to the catalytic domain through a disulfide bridge and is opposite to the active site. There are three disulfide bonds, and the C-terminus contains helical structures that run across the N-terminus of the β-barrel. The catalytic domain contains a calcium binding site that exposes the <scene name='Factor_IX/Rfnscene_5/1'>148-loop</scene> for proteolytic cleavage. This calcium ion is stabilized by Glu-70, Glu-77, Glu-80 and a main chain oxygens of Asn-72 and Glu-75. This site seems to be preformed, unlike the calcium binding sites that are generated in the Gla domain upon calcium binding.