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| | ==Crystal structure of prothrombin deletion mutant residues 146-167 ( Form II ).== | | ==Crystal structure of prothrombin deletion mutant residues 146-167 ( Form II ).== |
| - | <StructureSection load='5edk' size='340' side='right' caption='[[5edk]], [[Resolution|resolution]] 3.21Å' scene=''> | + | <StructureSection load='5edk' size='340' side='right'caption='[[5edk]], [[Resolution|resolution]] 3.21Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[5edk]] is a 1 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5EDK OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5EDK FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[5edk]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5EDK OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5EDK FirstGlance]. <br> |
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene>, <scene name='pdbligand=NAG:N-ACETYL-D-GLUCOSAMINE'>NAG</scene></td></tr> | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 3.214Å</td></tr> |
| - | <tr id='NonStdRes'><td class="sblockLbl"><b>[[Non-Standard_Residue|NonStd Res:]]</b></td><td class="sblockDat"><scene name='pdbligand=CGU:GAMMA-CARBOXY-GLUTAMIC+ACID'>CGU</scene></td></tr> | + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CGU:GAMMA-CARBOXY-GLUTAMIC+ACID'>CGU</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene>, <scene name='pdbligand=NAG:N-ACETYL-D-GLUCOSAMINE'>NAG</scene></td></tr> |
| - | <tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[4o03|4o03]]</td></tr>
| + | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=5edk FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5edk OCA], [https://pdbe.org/5edk PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5edk RCSB], [https://www.ebi.ac.uk/pdbsum/5edk PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5edk ProSAT]</span></td></tr> |
| - | <tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Thrombin Thrombin], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.4.21.5 3.4.21.5] </span></td></tr>
| + | |
| - | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5edk FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5edk OCA], [http://pdbe.org/5edk PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5edk RCSB], [http://www.ebi.ac.uk/pdbsum/5edk PDBsum]</span></td></tr> | + | |
| | </table> | | </table> |
| | == Disease == | | == Disease == |
| - | [[http://www.uniprot.org/uniprot/THRB_HUMAN THRB_HUMAN]] Defects in F2 are the cause of factor II deficiency (FA2D) [MIM:[http://omim.org/entry/613679 613679]]. It is a very rare blood coagulation disorder characterized by mucocutaneous bleeding symptoms. The severity of the bleeding manifestations correlates with blood factor II levels.<ref>PMID:14962227</ref> <ref>PMID:6405779</ref> <ref>PMID:3771562</ref> <ref>PMID:3567158</ref> <ref>PMID:3801671</ref> <ref>PMID:3242619</ref> <ref>PMID:2719946</ref> <ref>PMID:1354985</ref> <ref>PMID:1421398</ref> <ref>PMID:1349838</ref> <ref>PMID:7865694</ref> <ref>PMID:7792730</ref> Genetic variations in F2 may be a cause of susceptibility to ischemic stroke (ISCHSTR) [MIM:[http://omim.org/entry/601367 601367]]; also known as cerebrovascular accident or cerebral infarction. A stroke is an acute neurologic event leading to death of neural tissue of the brain and resulting in loss of motor, sensory and/or cognitive function. Ischemic strokes, resulting from vascular occlusion, is considered to be a highly complex disease consisting of a group of heterogeneous disorders with multiple genetic and environmental risk factors.<ref>PMID:15534175</ref> Defects in F2 are the cause of thrombophilia due to thrombin defect (THPH1) [MIM:[http://omim.org/entry/188050 188050]]. It is a multifactorial disorder of hemostasis characterized by abnormal platelet aggregation in response to various agents and recurrent thrombi formation. Note=A common genetic variation in the 3-prime untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increased risk of venous thrombosis. Defects in F2 are associated with susceptibility to pregnancy loss, recurrent, type 2 (RPRGL2) [MIM:[http://omim.org/entry/614390 614390]]. A common complication of pregnancy, resulting in spontaneous abortion before the fetus has reached viability. The term includes all miscarriages from the time of conception until 24 weeks of gestation. Recurrent pregnancy loss is defined as 3 or more consecutive spontaneous abortions.<ref>PMID:11506076</ref> | + | [https://www.uniprot.org/uniprot/THRB_HUMAN THRB_HUMAN] Defects in F2 are the cause of factor II deficiency (FA2D) [MIM:[https://omim.org/entry/613679 613679]. It is a very rare blood coagulation disorder characterized by mucocutaneous bleeding symptoms. The severity of the bleeding manifestations correlates with blood factor II levels.<ref>PMID:14962227</ref> <ref>PMID:6405779</ref> <ref>PMID:3771562</ref> <ref>PMID:3567158</ref> <ref>PMID:3801671</ref> <ref>PMID:3242619</ref> <ref>PMID:2719946</ref> <ref>PMID:1354985</ref> <ref>PMID:1421398</ref> <ref>PMID:1349838</ref> <ref>PMID:7865694</ref> <ref>PMID:7792730</ref> Genetic variations in F2 may be a cause of susceptibility to ischemic stroke (ISCHSTR) [MIM:[https://omim.org/entry/601367 601367]; also known as cerebrovascular accident or cerebral infarction. A stroke is an acute neurologic event leading to death of neural tissue of the brain and resulting in loss of motor, sensory and/or cognitive function. Ischemic strokes, resulting from vascular occlusion, is considered to be a highly complex disease consisting of a group of heterogeneous disorders with multiple genetic and environmental risk factors.<ref>PMID:15534175</ref> Defects in F2 are the cause of thrombophilia due to thrombin defect (THPH1) [MIM:[https://omim.org/entry/188050 188050]. It is a multifactorial disorder of hemostasis characterized by abnormal platelet aggregation in response to various agents and recurrent thrombi formation. Note=A common genetic variation in the 3-prime untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increased risk of venous thrombosis. Defects in F2 are associated with susceptibility to pregnancy loss, recurrent, type 2 (RPRGL2) [MIM:[https://omim.org/entry/614390 614390]. A common complication of pregnancy, resulting in spontaneous abortion before the fetus has reached viability. The term includes all miscarriages from the time of conception until 24 weeks of gestation. Recurrent pregnancy loss is defined as 3 or more consecutive spontaneous abortions.<ref>PMID:11506076</ref> |
| | == Function == | | == Function == |
| - | [[http://www.uniprot.org/uniprot/THRB_HUMAN THRB_HUMAN]] Thrombin, which cleaves bonds after Arg and Lys, converts fibrinogen to fibrin and activates factors V, VII, VIII, XIII, and, in complex with thrombomodulin, protein C. Functions in blood homeostasis, inflammation and wound healing.<ref>PMID:2856554</ref> | + | [https://www.uniprot.org/uniprot/THRB_HUMAN THRB_HUMAN] Thrombin, which cleaves bonds after Arg and Lys, converts fibrinogen to fibrin and activates factors V, VII, VIII, XIII, and, in complex with thrombomodulin, protein C. Functions in blood homeostasis, inflammation and wound healing.<ref>PMID:2856554</ref> |
| | <div style="background-color:#fffaf0;"> | | <div style="background-color:#fffaf0;"> |
| | == Publication Abstract from PubMed == | | == Publication Abstract from PubMed == |
| - | The zymogen prothrombin is proteolytically converted by factor Xa to the active protease thrombin in a reaction that is accelerated >3,000-fold by cofactor Va. This physiologically important effect is paradigmatic of analogous cofactor-dependent reactions in the coagulation and complement cascades, but its structural determinants remain poorly understood. Prothrombin has three linkers connecting the N-terminal Gla domain to kringle-1 (Lnk1), the two kringles (Lnk2), and kringle-2 to the C-terminal protease domain (Lnk3). Recent developments indicate that the linkers, and particularly Lnk2, confer on the zymogen significant flexibility in solution and enable prothrombin to sample alternative conformations. The role of this flexibility in the context of prothrombin activation was tested with several deletions. Removal of Lnk2 in almost its entirety (ProTDelta146-167) drastically reduces the enhancement of thrombin generation by cofactor Va from >3,000-fold to 60-fold because of a significant increase in the rate of activation in the absence of cofactor. Deletion of Lnk2 mimics the action of cofactor Va and offers insights into how prothrombin is activated at the molecular level. The crystal structure of ProTDelta146-167 reveals a contorted architecture where the domains are not vertically stacked, kringle-1 comes within 9 A of the protease domain, and the Gla-domain primed for membrane binding comes in contact with kringle-2. These findings broaden our molecular understanding of a key reaction of the blood coagulation cascade where cofactor Va enhances activation of prothrombin by factor Xa by compressing Lnk2 and morphing prothrombin into a conformation similar to the structure of ProTDelta146-167.
| + | A flexible linker (Lnk2) composed of 26 amino acids connects kringle-1 to kringle-2 in the coagulation factor prothrombin. Recent studies point to Lnk2 as a key determinant of the structure and function of this zymogen. Using a combination of mutagenesis, structural biology and single molecule spectroscopy we show how Lnk2 influences activation and conformational plasticity of prothrombin. Scrambling the sequence of Lnk2 is inconsequential on activation, and so is extension by as many as 22 residues. On the other hand, below a critical length of 15 residues, the rate of prothrombin activation increases (10-fold) in the absence of cofactor Va, and decreases (3-fold) in the presence of cofactor. Furthermore, activation by prothrombinase takes place without preference along the prethrombin-2 (cleavage at R271 first) or meizothrombin (cleavage at R320 first) pathways. Notably, these transitions in the rate and pathway of activation require the presence of phospholipids, pointing to an important physiological role for Lnk2 when prothrombin is anchored to the membrane. Two new crystal structures of prothrombin lacking 22 (ProTDelta146-167) or 14 (ProTDelta154-167) residues of Lnk2 document striking conformational rearrangements of domains located across this linker. FRET measurements of freely diffusing single molecules prove that these structural transitions are genuine properties of the zymogen in solution. These findings support a molecular model of prothrombin activation where Lnk2 presents the sites of cleavage at R271 and R320 to factor Xa in different orientations by pivoting the C-terminal kringle-2/protease domain pair on the N-terminal Gla domain/kringle-1 pair anchored to the membrane. |
| | | | |
| - | The linker connecting the two kringles plays a key role in prothrombin activation.,Pozzi N, Chen Z, Pelc LA, Shropshire DB, Di Cera E Proc Natl Acad Sci U S A. 2014 May 27;111(21):7630-5. doi:, 10.1073/pnas.1403779111. Epub 2014 May 12. PMID:24821807<ref>PMID:24821807</ref>
| + | How the Linker Connecting the Two Kringles Influences Activation and Conformational Plasticity of Prothrombin.,Pozzi N, Chen Z, Di Cera E J Biol Chem. 2016 Jan 12. pii: jbc.M115.700401. PMID:26763231<ref>PMID:26763231</ref> |
| | | | |
| | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> |
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| | __TOC__ | | __TOC__ |
| | </StructureSection> | | </StructureSection> |
| - | [[Category: Thrombin]] | + | [[Category: Homo sapiens]] |
| - | [[Category: Cera, E Di]] | + | [[Category: Large Structures]] |
| - | [[Category: Chen, Z]] | + | [[Category: Chen Z]] |
| - | [[Category: Pozzi, N]] | + | [[Category: Di Cera E]] |
| - | [[Category: Coagulation factor]] | + | [[Category: Pozzi N]] |
| - | [[Category: Enzyme mechanism]]
| + | |
| - | [[Category: Hydrolase]]
| + | |
| - | [[Category: Kinetic]]
| + | |
| - | [[Category: Kringle]]
| + | |
| - | [[Category: Protease]]
| + | |
| - | [[Category: Prothrombin]]
| + | |
| - | [[Category: Structure-function]]
| + | |
| Structural highlights
Disease
THRB_HUMAN Defects in F2 are the cause of factor II deficiency (FA2D) [MIM:613679. It is a very rare blood coagulation disorder characterized by mucocutaneous bleeding symptoms. The severity of the bleeding manifestations correlates with blood factor II levels.[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] Genetic variations in F2 may be a cause of susceptibility to ischemic stroke (ISCHSTR) [MIM:601367; also known as cerebrovascular accident or cerebral infarction. A stroke is an acute neurologic event leading to death of neural tissue of the brain and resulting in loss of motor, sensory and/or cognitive function. Ischemic strokes, resulting from vascular occlusion, is considered to be a highly complex disease consisting of a group of heterogeneous disorders with multiple genetic and environmental risk factors.[13] Defects in F2 are the cause of thrombophilia due to thrombin defect (THPH1) [MIM:188050. It is a multifactorial disorder of hemostasis characterized by abnormal platelet aggregation in response to various agents and recurrent thrombi formation. Note=A common genetic variation in the 3-prime untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increased risk of venous thrombosis. Defects in F2 are associated with susceptibility to pregnancy loss, recurrent, type 2 (RPRGL2) [MIM:614390. A common complication of pregnancy, resulting in spontaneous abortion before the fetus has reached viability. The term includes all miscarriages from the time of conception until 24 weeks of gestation. Recurrent pregnancy loss is defined as 3 or more consecutive spontaneous abortions.[14]
Function
THRB_HUMAN Thrombin, which cleaves bonds after Arg and Lys, converts fibrinogen to fibrin and activates factors V, VII, VIII, XIII, and, in complex with thrombomodulin, protein C. Functions in blood homeostasis, inflammation and wound healing.[15]
Publication Abstract from PubMed
A flexible linker (Lnk2) composed of 26 amino acids connects kringle-1 to kringle-2 in the coagulation factor prothrombin. Recent studies point to Lnk2 as a key determinant of the structure and function of this zymogen. Using a combination of mutagenesis, structural biology and single molecule spectroscopy we show how Lnk2 influences activation and conformational plasticity of prothrombin. Scrambling the sequence of Lnk2 is inconsequential on activation, and so is extension by as many as 22 residues. On the other hand, below a critical length of 15 residues, the rate of prothrombin activation increases (10-fold) in the absence of cofactor Va, and decreases (3-fold) in the presence of cofactor. Furthermore, activation by prothrombinase takes place without preference along the prethrombin-2 (cleavage at R271 first) or meizothrombin (cleavage at R320 first) pathways. Notably, these transitions in the rate and pathway of activation require the presence of phospholipids, pointing to an important physiological role for Lnk2 when prothrombin is anchored to the membrane. Two new crystal structures of prothrombin lacking 22 (ProTDelta146-167) or 14 (ProTDelta154-167) residues of Lnk2 document striking conformational rearrangements of domains located across this linker. FRET measurements of freely diffusing single molecules prove that these structural transitions are genuine properties of the zymogen in solution. These findings support a molecular model of prothrombin activation where Lnk2 presents the sites of cleavage at R271 and R320 to factor Xa in different orientations by pivoting the C-terminal kringle-2/protease domain pair on the N-terminal Gla domain/kringle-1 pair anchored to the membrane.
How the Linker Connecting the Two Kringles Influences Activation and Conformational Plasticity of Prothrombin.,Pozzi N, Chen Z, Di Cera E J Biol Chem. 2016 Jan 12. pii: jbc.M115.700401. PMID:26763231[16]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Wang W, Fu Q, Zhou R, Wu W, Ding Q, Hu Y, Wang X, Wang H, Wang Z. Prothrombin Shanghai: hypoprothrombinaemia caused by substitution of Gla29 by Gly. Haemophilia. 2004 Jan;10(1):94-7. PMID:14962227
- ↑ Board PG, Shaw DC. Determination of the amino acid substitution in human prothrombin type 3 (157 Glu leads to Lys) and the localization of a third thrombin cleavage site. Br J Haematol. 1983 Jun;54(2):245-54. PMID:6405779
- ↑ Rabiet MJ, Furie BC, Furie B. Molecular defect of prothrombin Barcelona. Substitution of cysteine for arginine at residue 273. J Biol Chem. 1986 Nov 15;261(32):15045-8. PMID:3771562
- ↑ Miyata T, Morita T, Inomoto T, Kawauchi S, Shirakami A, Iwanaga S. Prothrombin Tokushima, a replacement of arginine-418 by tryptophan that impairs the fibrinogen clotting activity of derived thrombin Tokushima. Biochemistry. 1987 Feb 24;26(4):1117-22. PMID:3567158
- ↑ Inomoto T, Shirakami A, Kawauchi S, Shigekiyo T, Saito S, Miyoshi K, Morita T, Iwanaga S. Prothrombin Tokushima: characterization of dysfunctional thrombin derived from a variant of human prothrombin. Blood. 1987 Feb;69(2):565-9. PMID:3801671
- ↑ Henriksen RA, Mann KG. Identification of the primary structural defect in the dysthrombin thrombin Quick I: substitution of cysteine for arginine-382. Biochemistry. 1988 Dec 27;27(26):9160-5. PMID:3242619
- ↑ Henriksen RA, Mann KG. Substitution of valine for glycine-558 in the congenital dysthrombin thrombin Quick II alters primary substrate specificity. Biochemistry. 1989 Mar 7;28(5):2078-82. PMID:2719946
- ↑ Miyata T, Aruga R, Umeyama H, Bezeaud A, Guillin MC, Iwanaga S. Prothrombin Salakta: substitution of glutamic acid-466 by alanine reduces the fibrinogen clotting activity and the esterase activity. Biochemistry. 1992 Aug 25;31(33):7457-62. PMID:1354985
- ↑ Morishita E, Saito M, Kumabashiri I, Asakura H, Matsuda T, Yamaguchi K. Prothrombin Himi: a compound heterozygote for two dysfunctional prothrombin molecules (Met-337-->Thr and Arg-388-->His). Blood. 1992 Nov 1;80(9):2275-80. PMID:1421398
- ↑ Iwahana H, Yoshimoto K, Shigekiyo T, Shirakami A, Saito S, Itakura M. Detection of a single base substitution of the gene for prothrombin Tokushima. The application of PCR-SSCP for the genetic and molecular analysis of dysprothrombinemia. Int J Hematol. 1992 Feb;55(1):93-100. PMID:1349838
- ↑ James HL, Kim DJ, Zheng DQ, Girolami A. Prothrombin Padua I: incomplete activation due to an amino acid substitution at a factor Xa cleavage site. Blood Coagul Fibrinolysis. 1994 Oct;5(5):841-4. PMID:7865694
- ↑ Degen SJ, McDowell SA, Sparks LM, Scharrer I. Prothrombin Frankfurt: a dysfunctional prothrombin characterized by substitution of Glu-466 by Ala. Thromb Haemost. 1995 Feb;73(2):203-9. PMID:7792730
- ↑ Casas JP, Hingorani AD, Bautista LE, Sharma P. Meta-analysis of genetic studies in ischemic stroke: thirty-two genes involving approximately 18,000 cases and 58,000 controls. Arch Neurol. 2004 Nov;61(11):1652-61. PMID:15534175 doi:61/11/1652
- ↑ Pihusch R, Buchholz T, Lohse P, Rubsamen H, Rogenhofer N, Hasbargen U, Hiller E, Thaler CJ. Thrombophilic gene mutations and recurrent spontaneous abortion: prothrombin mutation increases the risk in the first trimester. Am J Reprod Immunol. 2001 Aug;46(2):124-31. PMID:11506076
- ↑ Glenn KC, Frost GH, Bergmann JS, Carney DH. Synthetic peptides bind to high-affinity thrombin receptors and modulate thrombin mitogenesis. Pept Res. 1988 Nov-Dec;1(2):65-73. PMID:2856554
- ↑ Pozzi N, Chen Z, Di Cera E. How the Linker Connecting the Two Kringles Influences Activation and Conformational Plasticity of Prothrombin. J Biol Chem. 2016 Jan 12. pii: jbc.M115.700401. PMID:26763231 doi:http://dx.doi.org/10.1074/jbc.M115.700401
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