Crystal Structure of the Michaelis Complex between Tissue-type Plasminogen Activator and Plasminogen Activators Inhibitor-1

Lihu Gong,Min Liu,Tu Zeng,Xiaoli Shi,Cai Yuan,Peter A Andreasen,Mingdong Huang

doi:10.1074/jbc.m115.677567

Abstract

Thrombosis is a leading cause of death worldwide. Recombinant tissue-type plasminogen activator (tPA) is the Food and Drug Administration-approved thrombolytic drug. tPA is rapidly inactivated by endogenous plasminogen activator inhibitor-1 (PAI-1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort. Precise details, with atomic resolution, of the molecular interactions between tPA and PAI-1 remain unknown despite previous extensive studies. Here, we report the crystal structure of the tPA·PAI-1 Michaelis complex, which shows significant differences from the structure of its urokinase-type plasminogen activator analogue, the uPA·PAI-1 Michaelis complex. The PAI-1 reactive center loop adopts a unique kinked conformation. The structure provides detailed interactions between tPA 37- and 60-loops with PAI-1. On the tPA side, the S2 and S1β pockets open up to accommodate PAI-1. This study provides structural basis to understand the specificity of PAI-1 and to design newer generation of thrombolytic agents with reduced PAI-1 inactivation.

Highlights

Recombinant tissue-type plasminogen activator is a potent fibrinolytic agent used in clinics and is inactivated by endogenous plasminogen activator inhibitor-1 (PAI-1)
The type plasminogen activator (tPA)-SPD domain is responsible for its plasminogen activation function and its inhibition by PAI-1
We generated three mutations in tPA-serine protease domain (tPA-SPD): S478A to render the tPA-SPD catalytically inactive, so the Michaelis complex does not proceed to the stable, covalent complex; N448Q to remove the glycosylation on tPASPD, increasing the homogeneity of the recombinant protein and facilitating protein crystallization; and C395A mutation to remove the disulfide bond linked to K2 domain (Fig. 1B)

Summary

Background

Recombinant tissue-type plasminogen activator (tPA) is a potent fibrinolytic agent used in clinics and is inactivated by endogenous PAI-1. A high dose of recombinant tPA (up to 100 mg/50 kg) is typically needed in clinical applications, in part because of the rapid inactivation of recombinant tPA by endogenous plasminogen activators inhibitor 1 (PAI-1 or SERPINE1), a member of serine protease inhibitor (SERPIN) family Such high dosage leads to dangerous side effects, e.g. intracranial hemorrhage and neurotoxicity [4, 5]. We determined the crystal structure of the Michaelis complex between tPA and PAI-1, which identifies key residues of tPA in the interface and provides insight on the specificity of PAI-1 for tPA and uPA. This structure explains the PAI-1 resisting property of tenecteplase and offers important clues to design newer generation of PAI-1-resistant tPA variants

Experimental Procedures

Results and Discussion

Multiplicity Ra merge