Factor IX: Difference between revisions

A [[pro-peptide]] functions as a recognition element for a vitamin K-dependent carboxylase (γ-glutamyl carboxylase) which modifies 12 glutamic acid residues to gamma-carboxyglutamyl (<scene name='Factor_IX/Ixstructure_residue/3'>Gla</scene>) residues<ref>PMID:12554099</ref>. These residues are required for the association with the anionic phospholipid surface through Ca2+-dependent binding. Additional details in [[Calcium ions the Gla domain]].

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 serine protease contains a catalytic triad of <scene name='Factor_IX/Ixstructure_catalytictriad/3'>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 serine proteases. The active FIXa, can then interact with its cofactor, FVIIIa, to form a membrane-bound Xase complex, which activated FX to FXa.

== γ-Carboxyglutamic Acid (Gla) Domain ==

   

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/3'>Gla domain</scene> is made up of 10-13 of γ-carboxyglutamic acid residues and requires both <scene name='Factor_IX/Gladomain_1/8'>Ca2+ and Mg2+ </scene> ions for membrane association and stabilization of its active three dimensional conformation <ref>PMID:3511981</ref>.  

This structure emphasizes the possible role of magnesium in Gla domain binding to membrane surfaces. However upon examination of the calcium bound Factor IX-(1-46) complex revealed that the calcium coordination within this FIX Gla domain structure differs from other vitamin K-dependent proteins (prothrombin, Factor VII, and Factor X). This deviation may be explained by the interaction of the snake venom FIX-bp interaction.

To further probe into the structure of FIX Gla domain, [[conformation-specific anti-Factor IX antibodies]] were utilized. Using an  

The Gla domain of Factor IX consists of an N-terminal loop and three short ''&omega;''-<scene name='Factor_IX/Antifactor_2/3'>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 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.

== Serine Protease Domain ==  

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 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.  

==3D structures of factor IX==

==Additional Resources==