1VR1

Specifity for Plasminogen Activator Inhibitor-1

Summary for 1VR1

• Entry DOI: 10.2210/pdb1vr1/pdb
• Descriptor: thrombin, Hirudin, ... (4 entities in total)
• Functional Keywords: thrombin, blood clotting-hydrolase inhibitor complex, blood clotting/hydrolase inhibitor
• Biological source: Homo sapiens (human); More
• Cellular location: Secreted, extracellular space: P00734 P00734; Secreted: P28504
• Total number of polymer chains: 3
• Total formula weight: 34738.76

Authors

Dekker, R.J.,Eichinger, A.,Stoop, A.A.,Bode, W.,Pannekoek, H.,Horrevoets, A.J.G. (deposition date: 1998-12-11, release date: 1999-12-29, Last modification date: 2024-10-09)

Primary citation

Dekker, R.J.,Eichinger, A.,Stoop, A.A.,Bode, W.,Pannekoek, H.,Horrevoets, A.J.G.
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
J.Mol.Biol., 293:613-627, 1999

PubMed Abstract: 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.

PubMed: 10543954
DOI: 10.1006/jmbi.1999.3178

Experimental method

X-RAY DIFFRACTION (1.9 Å)