1vr1
Specifity for Plasminogen Activator Inhibitor-1
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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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