X-ray Crystal Structures and NMR Structures of Tissue Factor, Factor VII, Factor VIIa, TFPI, and antibodies to these proteins
A wealth of structural information is now available for coagulation factor VII, tissue factor (TF), and the Kunitz-type inhibitor, tissue factor pathway inhibitor (TFPI). (Note: TF is also sometimes called thromboplastin, tissue thromboplastin, coagulation factor III, or CD142. The locus of the tissue factor gene is sometimes symbolized by F3 or CF3.) Click here for a description of the domain organization of TF, factor VIIa and TFPI. Below are brief descriptions of published x-ray and/or NMR structures of these proteins, followed in each section by a table listing the relevant structures with links to their corresponding coordinate files at the Protein Data Bank (PDB). At the bottom of this page is a description of how to view these structures on your personal computer.
TF was the March 2006 molecule of the month at the Protein Data Bank (PDB).
Tissue Factor
Human tissue factor is an integral membrane protein consisting of an extracellular domain of 217 or 219 amino acids, a single membrane-spanning domain, and a short cytoplasmic tail. The isolated extracellular domain can be produced by recombinant means in bacteria and other expression systems. This form of the protein, which apparently does not occur naturally, is highly water soluble and has therefore been called soluble tissue factor (sTF). sTF is very useful because it permits biochemical and biophysical studies of tissue factor function in both the presence and absence of phospholipid surfaces (reviewed by Morrissey [1].). In 1994, the X-ray crystal structure of human sTF was solved by two groups, to a resolution of 2.2 [2] or 2.4 Angstroms [3]. Tissue factor was the first protein cofactor of the blood clotting system whose three-dimensional structure was solved. It is a member of the class 2 cytokine receptor superfamily and is composed of two fibronectin type-III domains connected to each other at an angle of about 120 degrees. Subsequent to the 1994 publications cited above, the Muller et al. structure was further refined to a resolution of 1.7 Angstroms [4]. More recently, the X-ray crystal structure of rabbit sTF has been solved [5]. The authors of this latter study found two molecules of rabbit sTF per unit cell, with different angles between the two fibronectin type III domains. They pointed out that the hinge region joining these two domains is apparently more flexible than had previously been realized.
| PDB entry | Description | Resolution |
| 1BOY | Human sTF | 2.20 A |
| 2HFT | Human sTF | 1.69 A |
| 1TFH | Human sTF | 2.40 A |
| 1A21 | Rabbit sTF (two structures) | 2.35 A |
| 2CEH | Human TF cytoplasmic domain | (NMR) |
| 2CEZ | Human TF cytoplasmic domain, phosphorylated at Ser253 | (NMR) |
| 2CFJ | Human TF cytoplasmic domain, phosphorylated at Ser258 | (NMR) |
| 2CEF | Human TF cytoplasmic domain, phosphorylated at Ser253 & Ser258 | (NMR) |
Tissue Factor-Antibody Complex
Listed here are X-ray crystal structures of human sTF bound to anti-TF monoclonal
antibodies (or Fab fragments) that do not interfere with factor VIIa binding but which inhibit factor X activation by the TF:VIIa complex.
The epitopes for these antibodies are thought to overlap a putative substrate binding site (exosite) on TF.
| PDB entry | Description | Resolution |
| 1AHW | Human sTF in complex with TF8-5G9 Fab | 3.00 A |
| 1FGN | TF8-5G9 Fab | 2.50 A |
| 1JPS | Human sTF in complex with humanized Fab D3h44 | 1.85 A |
| 1JPT | Fab D3H44 | 1.85 A |
| 1K6Q | Fab fragment D3 | 2.40 A |
| 1UJ3 | Humanized Fab fragment of antiTF antibody in complex with sTF | 2.10 A |
| 1PG7 | Murine 6A6 Fab in complex with humanized anti-TF D3H44 Fab | 2.50 A |
Tissue Factor-Factor VIIa Complex
The ligand for tissue factor is the serine protease, factor VIIa. In
1996, Banner et al. [6] solved the X-ray crystal structure
of active site-inhibited human factor VIIa (sometimes called factor VIIai)
bound to a protease-cleaved form of human sTF.
The VIIa inhibitor was a tripeptidyl-chloromethylketone. This was the first time that the three-dimensional structure
of the complex of a blood coagulation protease with its cognate protein
cofactor was solved. It is also remarkable in that all of the domains
of factor VIIa (including the Gla domain) were well resolved. Since then, several more structures of the sTF-VIIa
complex have been solved, typically with a protease inhibitor in the active site of factor VIIa. Since TF:VIIa occupies a priveleged position as the enzyme that initiates the blood clotting cascade, it is an attractive target for the development of novel anti-thrombotic drugs.
| PDB entry | Description | Resolution |
| 1DAN | Human sTF in complex with factor VIIa, inhibited by D-Phe-Phe-Arg chloromethylketone | 2.00 A |
| 1FAK | Human sTF in complex with factor VIIa, inhibited by modified BPTI | 2.10 A |
| 1J9C | Human sTF in complex with factor VIIa, inhibited by D-Phe-Phe-Arg | 2.90 A |
| 1W0Y | Human sTF in complex with factor VIIa, inhibited by a phenylglycine-based inhibitor | 2.50 A |
| 1W2K | Human sTF in complex with factor VIIa, inhibited by a phenylglycine-based inhibitor | 3.00 A |
| 1WQV | Human sTF in complex with factor VIIa, inhibited by a peptide mimetic inhibitor | 2.50 A |
| 1WSS | Human sTF in complex with factor VIIa, inhibited by a peptide mimetic inhibitor | 2.60 A |
| 1WTG | Human sTF in complex with factor VIIa, inhibited by a peptide mimetic inhibitor | 2.20 A |
| 1WUN | Human sTF in complex with factor VIIa, inhibited by a peptide mimetic inhibitor | 2.40 A |
| 1WV7 | Human sTF in complex with factor VIIa, inhibited by a peptide mimetic inhibitor | 2.70 A |
| 1Z6J | Human sTF in complex with factor VIIa, inhibited by a substituted pyrazinone-based inhibitor | 2.00 A |
| 2B7D | Human sTF in complex with factor VIIa, inhibited by a small molecule inhibitor | 2.24 A |
| 2FLR | Human sTF in complex with factor VIIa, inhibited by a 5-azaindole inhibitor | 2.35 A |
| 2AEI | Human sTF in complex with factor VIIa, inhibited by a fluoropyridine-based inhibitor | 2.52 A |
| 2C4F | Human sTF in complex with factor VIIa, inhibited by a fluoropyridine-based inhibitor | 1.72 A |
| 2F9B | Human sTF in complex with factor VIIa, inhibited by a heterocyclic inhibitor | 2.54 A |
| 2PUQ | Human sTF in complex with factor VIIa, inhibited by D-Trp-Tyr-Thr-Arg chloromethylketone | 2.05 A |
| 2A2Q | Human sTF in complex with factor VIIa, inhibited by p-aminobenzamidine; also has sodium, zinc and magnesium ions bound to factor VIIa (in addition to calcium ions) | 1.80 A |
| 2AER | Human sTF in complex with factor VIIa, inhibited by benzamidine; also has sodium, zinc and magnesium ions bound to factor VIIa (in addition to calcium ions) | 1.87 A |
| 2FIR | Human sTF in complex with factor VIIa, inhibited by D-Phe-Pro-Arg chloromethylketone; also has sodium, zinc and magnesium ions bound to factor VIIa (in addition to calcium ions) | 2.00 A |
| 2B8O | Human sTF in complex with factor VIIa, inhibited by Glu-Gly-Arg chloromethylketone; also has sodium, zinc and magnesium ions bound to factor VIIa (in addition to calcium ions) | 2.80 A |
| 3TH2 | Human sTF in complex with factor VIIa, inhibited by benzamidine; also has sodium and magnesium ions bound to factor VIIa (in addition to calcium ions) | 1.72 A |
| 3TH3 | Human sTF in complex with factor VIIa, inhibited by dansyl-Glu-Gly-Arg-chloromethylketone | 2.70A |
| 3TH4 | Human sTF in complex with factor VIIa, inhibited by dansyl-Glu-Gly-Arg-chloromethylketone; also has sodium and magnesium ions bound to factor VIIa (in addition to calcium ions) | 1.80 A |
| 1O5D | Human sTF in complex with factor VIIa, inhibited by a synthetic uPA inhibitor | 2.05 A |
| 2FLB | Human sTF in complex with factor VIIa, inhibited by a novel hydroxy pyrazole based factor IXa Iinhibitor | 1.95 A |
| 2EC9 | Human sTF in complex with factor VIIa, inhibited by BCX-3607 | 2.00 A |
| 2ZP0 | Human sTF in complex with factor VIIa, inhibited by benzylsulfonamide-D-Ile-Gln-p-aminobenzamidine | 2.70 A |
| 3ELA | Human sTF in complex with mutant factor VIIa (V158D,E296V,M298Q), inhibited by PhePheArg-chloromethylketone | 2.20 A |
| 2ZWL | Human sTF in complex with factor VIIa, inhibited by a selective peptide inhibitor | 2.20 A |
| 2ZZU | Human sTF in complex with factor VIIa, inhibited by ethylsulfonamide-D-5-(3-carboxybenzyloxy)-Trp-Gln-p-aminobenzamidine | 2.50 A |
Free Factor VII and VIIa
Factor VII is the zymogen (inactive precursor), while factor VIIa
is the active enzyme (serine protease). Zymogen factor VII (a single-chain protein),
is converted to factor VIIa (a two-chain protein) by proteolysis of a
single Arg-Ile peptide bond. The two polypeptide chains of factor VIIa
are held together by a disulfide bond. In human plasma, about 99% of factor VII circulates as the zymogen, while about 1% circulates
as the active enzyme, factor VIIa [9]. Structures for both free factor VIIa and zymogen factor VII have been reported. In addition, X-ray and neutron scattering
studies of human factor VIIa in solution have shown that this protein has an extended
or elongated domain structure in the absence of tissue factor [7].
Finally, a computer model of the solution structure of the light chain
of human factor VIIa has been published [8], which
also predicts that factor VIIa has an extended conformation in solution.
| PDB entry | Definition | Resolution |
| 1JBU | Human Factor VII zymogen (lacking the Gla domain and first EGF domain), with an exosite peptide inhibitor bound | 2.00 A |
| 1QFK | Human Gla-domainless factor VIIa, inhibited by D-Phe-Phe-Arg chloromethylketone | 2.80 A |
| 1CVW | Human Gla-domainless factor VIIa, inhibited by dansyl-Glu-Gly-Arg chloromethylketone | 2.28 A |
| 1DVA | Human Gla-domainless factor VIIa, in complex with peptide exosite inhibitor E-76 | 3.00 A |
| 1KLI | Human factor VIIa (lacking the Gla domain and first EGF domain), no benzamidine (also has ethylene glycol) | 1.69 A |
| 1KLJ | Human factor VIIa (lacking the Gla domain and first EGF domain), inhibited by benzamidine (also has ethylene glycol) | 2.44 |
| 1YGC | Human factor VIIa (lacking the Gla domain and first EGF domain), inhibited by active site inhibitor G17905 | 2.00 A |
| 1W7X | Human factor VIIa (lacking the Gla domain and first EGF domain), inhibited by a phenylglycine-based inhibitor | 1.80 A |
| 1W8B | Human factor VIIa (lacking the Gla domain and first EGF domain), inhibited by a phenylglycine-based inhibitor | 3.00 A |
| 2BZ6 | Human factor VIIa (lacking the Gla domain and first EGF domain), inhibited by an orally available, small molecule inhibitor | 1.60 A |
| 4ISI | Human factor VIIa (lacking the Gla domain and first EGF domain), inhibited by (6S)-N-(4-CARBAMIMIDOYLBENZYL)-1-CHLORO-3-(CYCLOBUTYLAMINO)-8,8-DIETHYL-4-OXO-4,6,7,8-TETRAHYDROPYRROLO[1,2-A]PYRAZINE-6-CARBOXAMIDE | 1.94 A |
| 4ISH | Human factor VIIa (lacking the Gla domain and first EGF domain), inhibited by BMS-593214 (2′-[(6R,6AR,11BR)-2-carbamimidoyl-6,6A,7,11B-tetrahydro-5H-indeno[2,1-C]quinolin-6-YL]-5′-hydroxy-4′-methoxybiphenyl-4-carboxylic acid) | 1.82 A |
| 4JYV | Human factor VIIa (lacking the Gla domain and first EGF domain), inhibited by (2R)-2-[3-ETHOXY-4-(PROPAN-2-YLOXY)PHENYL]-2-(ISOQUINOLIN-6-YLAMINO)-N-[(3-SULFAMOYLPHENYL)SULFONYL]ETHANAMIDE | 2.19 A |
| 4JZD | Human factor VIIa (lacking the Gla domain and first EGF domain), inhibited by 2-{2-[(4-carbamimidoylphenyl)carbamoyl]-6-methoxypyridin-3-yl}-5-{[(2S)-1-hydroxy-3,3-dimethylbutan-2-yl]carbamoyl}benzoic acid | 2.20 A |
| 4JZE | Human factor VIIa (lacking the Gla domain and first EGF domain), inhibited by 2-{2-[(1-aminoisoquinolin-6-yl)carbamoyl]-6-methoxypyridin-3-yl}-5-{[(2S)-1-hydroxy-3,3-dimethylbutan-2-yl]carbamoyl}benzoic acid | 1.52 A |
| 4JZF | Human factor VIIa (lacking the Gla domain and first EGF domain), inhibited by 2-{2-[(3-carbamoylphenyl)carbamoyl]-6-methoxypyridin-3-yl}-5-{[(2S)-1-hydroxy-3,3-dimethylbutan-2-yl]carbamoyl}benzoic acid | 1.84 A |
| 1F7E | Human factor VII, first EGF domain (-fucose) | (NMR) |
| 1F7M | Human factor VII, first EGF domain (-fucose) | (NMR) |
| 1FF7 | Human factor VII, first EGF domain (+fucose) | (NMR) |
| 1FFM | Human factor VII, first EGF domain (+fucose) | (NMR) |
| 1BF9 | Human factor VII, first EGF domain (-calcium) | (NMR) |
Tissue Factor Pathway Inhibitor (TFPI) and TFPI-2
TFPI contains three Kunitz-type protease inhibitor domains. The first
Kunitz domain reacts with the active site of factor VIIa, while the second
Kunitz domain reacts with the active site of factor Xa.
| PDB entry | Description | Resolution |
| 1ADZ | Human TFPI (second Kunitz domain) | (NMR; 30 structures) |
| 1TFX | Human TFPI (second Kunitz domain) in complex with porcine trypsin | 2.60 A |
| 1IRH | Human TFPI (third Kunitz domain) | (NMR) |
| 1ZR0 | Human TFPI-2 (first Kunitz domain) in complex with bovine trypsin | 1.80 A |
| 4DTG | Human TFPI (second Kunitz domain) complexed with Fab fragment of anti-TFPI monoclonal antibody mAb 2021 | 1.80 A |
How to view these structures
These protein structures can be viewed within the PDB’s
World-Wide Web site using various web-based viewing options. Alternatively, you can download the coordinates for each structure to your own computer. Several
free programs are available for viewing 3-D structures on your personal computer, including the following:
- RasMol
(free download) viewer. This site also has tutorials for using RasMol and
Chime, as well as links to sites for other viewers . - RasTop (free download) is an enhanced version of RasMol with an expanded user interface. It allows you to view multiple structures together. One of the most useful features is that it allows you to export POV-Ray scripts.
- Deep View Swiss-Pdb Viewer
(free download). A very feature-rich viewer that also allows you to view several structures together and also to manipulate structures. It outputs POV-Ray scripts, allowing high resolution images of proteins (including ribbon images, which RasTop does not export into POV-Ray scripts). - POV-Ray (free download) accepts special scripts generated by RasTop, Deep View, Discovery Studio Visualizer and others that will allow you to produce beautiful, high resolution images of proteins. Highly recommended.
- UC Berkeley enhanced version of RasMol (free download), allows you to view two structures together in the same window.
- Chime (free download) is a very useful web browser plug-in for viewing 3-D structures.
- Discovery Studio Visualizer (formerly ViewerPro) from Accelrys Software (free download) is a feature-rich viewer.
References
1. Morrissey JH (1995). Tissue factor modulation of factor VIIa activity: Use in measuring trace levels of factor VIIa in plasma. Thromb. Haemost. 74:185-188. [PubMed]
2. Harlos K, Martin DMA, O’Brien DP, Jones EY, Stuart DI, Polikarpov I, Miller A, Tuddenham EGD, Boys CWG (1994). Crystal structure of the extracellular region of human tissue factor. Nature 370:662-666. [PubMed]
3. Muller YA, Ultsch MH, Kelley RF, De Vos AM (1994). Structure of the extracellular domain of human tissue factor: Location of the factor VIIa binding site. Biochemistry 33:10864-10870. [PubMed]
4. Muller YA, Ultsch MH, de Vos AM (1996). The crystal structure of the extracellular domain of human tissue factor refined to 1.7 A resolution. J. Molec. Biol. 256:144-159. [PubMed]
5. Muller YA, Kelley RF, de Vos AM (1998) Hinge bending within the cytokine receptor superfamily revealed by the 2.4 A crystal structure of the extracellular domain of rabbit tissue factor. Protein Sci 7:1106-1115. [PubMed]
6. Banner DW, D’Arcy A, Chene C, Winkler FK, Guha A, Konigsberg WH, Nemerson Y, Kirchhofer D (1996). The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor. Nature 380:41-46. [PubMed]
7. Ashton AW, Boehm MK, Johnson DJ, Kemball-Cook G, Perkins SJ (1998) The solution structure of human coagulation factor VIIa in its complex with tissue factor is similar to free factor VIIa: A study of a heterodimeric receptor-ligand complex by X-ray and neutron scattering and computational modeling. Biochemistry 37:8208-8217. [PubMed]
8. Perera L, Darden TA, Pedersen LG (1999) Probing the structural changes in the light chain of human coagulation factor VIIa due to tissue factor association. Biophys J 77:99-113. [PubMed]
9. Morrissey JH, Macik BG, Neuenschwander PF, Comp PC (1993) Quantitation of activated factor VII levels in plasma using a tissue factor mutant selectively deficient in promoting factor VII activation. Blood 81:734-744. [PubMed]