1vr1: Difference between revisions
New page: left|200px<br /> <applet load="1vr1" size="450" color="white" frame="true" align="right" spinBox="true" caption="1vr1, resolution 1.9Å" /> '''Specifity for Plasmi... |
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[[Image:1vr1.gif|left|200px]]<br /> | [[Image:1vr1.gif|left|200px]]<br /><applet load="1vr1" size="350" color="white" frame="true" align="right" spinBox="true" | ||
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caption="1vr1, resolution 1.9Å" /> | caption="1vr1, resolution 1.9Å" /> | ||
'''Specifity for Plasminogen Activator Inhibitor-1'''<br /> | '''Specifity for Plasminogen Activator Inhibitor-1'''<br /> | ||
==Overview== | ==Overview== | ||
Substitution of the native variable region-1 (VR1/37-loop) of thrombin by | Substitution of the native variable region-1 (VR1/37-loop) of thrombin by the corresponding VR1 of tissue-type plasminogen activator (thrombin-VR1(tPA)) increases the rate of inhibition by plasminogen activator inhibitor type 1 (PAI-1) by three orders of magnitude, and is thus sufficient to confer PAI-1 specificity to a heterologous serine protease. A structural and kinetical approach to establish the function of the VR1 loop of t-PA in the context of the thrombin-VR1(tPA) variant is described. The crystal structure of thrombin-VR1(tPA) was resolved and showed a conserved overall alpha-thrombin structure, but a partially disordered VR1 loop as also reported for t-PA. The contribution of a prominent charge substitution close to the active site was studied using charge neutralization variants thrombin-E39Q(c39) and thrombin-VR1(tPA)-R304Q(c39), resulting in only fourfold changes in the PAI-1 inhibition rate. Surface plasmon resonance revealed that the affinity of initial reversible complex formation between PAI-1 and catalytically inactive Ser195-->Ala variants of thrombin and thrombin-VR1(tPA) is only increased fivefold, i.e. KD is 652 and 128 nM for thrombin-S195A and thrombin-S195A-VR1(tPA), respectively. We established that the partition ratio of the suicide substrate reaction between the proteases and PAI-1 was largely unaffected in any variant studied. Hirugen allosterically decreases the rate of thrombin inhibition by PAI-1 2.5-fold and of thrombin-VR1(tPA) 20-fold, by interfering with a unimolecular step in the reaction, not by decreasing initial complex formation or by altering the stoichiometry. Finally, kinetic modeling demonstrated that acylation is the rate-limiting step in thrombin inhibition by PAI-1 (k approximately 10(-3) s(-1)) and this kinetic block is alleviated by the introduction of the tPA-VR1 into thrombin (k>1 s(-1)). We propose that the length, flexibility and different charge architecture of the VR1 loop of t-PA invoke an induced fit of the reactive center loop of PAI-1, thereby enhancing the rate of acylation in the Michaelis complex between thrombin-VR1(t-PA) and PAI-1 by more than two orders of magnitude. | ||
==Disease== | ==Disease== | ||
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==About this Structure== | ==About this Structure== | ||
1VR1 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens]. Full crystallographic information is available from [http:// | 1VR1 is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1VR1 OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Bode, W.]] | [[Category: Bode, W.]] | ||
[[Category: Dekker, R | [[Category: Dekker, R J.]] | ||
[[Category: Eichinger, A.]] | [[Category: Eichinger, A.]] | ||
[[Category: Horrevoets, A | [[Category: Horrevoets, A J.G.]] | ||
[[Category: Pannekoek, H.]] | [[Category: Pannekoek, H.]] | ||
[[Category: Stoop, A | [[Category: Stoop, A A.]] | ||
[[Category: thrombin]] | [[Category: thrombin]] | ||
''Page seeded by [http:// | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 15:37:46 2008'' | ||
Revision as of 16:37, 21 February 2008
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Specifity for Plasminogen Activator Inhibitor-1
OverviewOverview
Substitution of the native variable region-1 (VR1/37-loop) of thrombin by the corresponding VR1 of tissue-type plasminogen activator (thrombin-VR1(tPA)) increases the rate of inhibition by plasminogen activator inhibitor type 1 (PAI-1) by three orders of magnitude, and is thus sufficient to confer PAI-1 specificity to a heterologous serine protease. A structural and kinetical approach to establish the function of the VR1 loop of t-PA in the context of the thrombin-VR1(tPA) variant is described. The crystal structure of thrombin-VR1(tPA) was resolved and showed a conserved overall alpha-thrombin structure, but a partially disordered VR1 loop as also reported for t-PA. The contribution of a prominent charge substitution close to the active site was studied using charge neutralization variants thrombin-E39Q(c39) and thrombin-VR1(tPA)-R304Q(c39), resulting in only fourfold changes in the PAI-1 inhibition rate. Surface plasmon resonance revealed that the affinity of initial reversible complex formation between PAI-1 and catalytically inactive Ser195-->Ala variants of thrombin and thrombin-VR1(tPA) is only increased fivefold, i.e. KD is 652 and 128 nM for thrombin-S195A and thrombin-S195A-VR1(tPA), respectively. We established that the partition ratio of the suicide substrate reaction between the proteases and PAI-1 was largely unaffected in any variant studied. Hirugen allosterically decreases the rate of thrombin inhibition by PAI-1 2.5-fold and of thrombin-VR1(tPA) 20-fold, by interfering with a unimolecular step in the reaction, not by decreasing initial complex formation or by altering the stoichiometry. Finally, kinetic modeling demonstrated that acylation is the rate-limiting step in thrombin inhibition by PAI-1 (k approximately 10(-3) s(-1)) and this kinetic block is alleviated by the introduction of the tPA-VR1 into thrombin (k>1 s(-1)). We propose that the length, flexibility and different charge architecture of the VR1 loop of t-PA invoke an induced fit of the reactive center loop of PAI-1, thereby enhancing the rate of acylation in the Michaelis complex between thrombin-VR1(t-PA) and PAI-1 by more than two orders of magnitude.
DiseaseDisease
Known diseases associated with this structure: Dysprothrombinemia OMIM:[176930], Hyperprothrombinemia OMIM:[176930], Hypoprothrombinemia OMIM:[176930]
About this StructureAbout this Structure
1VR1 is a Single protein structure of sequence from Homo sapiens. Full crystallographic information is available from OCA.
ReferenceReference
The variable region-1 from tissue-type plasminogen activator confers specificity for plasminogen activator inhibitor-1 to thrombin by facilitating catalysis: release of a kinetic block by a heterologous protein surface loop., Dekker RJ, Eichinger A, Stoop AA, Bode W, Pannekoek H, Horrevoets AJ, J Mol Biol. 1999 Oct 29;293(3):613-27. PMID:10543954
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