The length of the reactive center loop modulates the latency transition of plasminogen activator inhibitor-1

Abstract

Plasminogen activator inhibitor-1 (PAI-1) belongs to the serine protease inhibitor (serpin) protein family, which has a common tertiary structure consisting of three beta-sheets and several alpha-helices. Despite the similarity of its structure with those of other serpins, PAI-1 is unique in its conformational lability, which allows the conversion of the metastable active form to a more stable latent conformation under physiological conditions. For the conformational conversion to occur, the reactive center loop (RCL) of PAI-1 must be mobilized and inserted into the major beta-sheet, A sheet. In an effort to understand how the structural conversion is regulated in this conformationally labile serpin, we modulated the length of the RCL of PAI-1. We show that releasing the constraint on the RCL by extension of the loop facilitates a conformational transition of PAI-1 to a stable state. Biochemical data strongly suggest that the stabilization of the transformed conformation is owing to the insertion of the RCL into A beta-sheet, as in the known latent form. In contrast, reducing the loop length drastically retards the conformational change. The results clearly show that the constraint on the RCL is a factor that regulates the conformational transition of PAI-1.

Figure 1.

A schematic diagram of PAI-1. (A) The native structure (Sharp et al. 1999; 1B3K.pdb). The β-sheets A is indicated as black strands, and the RCL and the first stand of β-sheet C (s1C) is indicated in light gray. The scissile peptide bond, recognized by tPA and uPA, is indicated with an arrow. Glu 350, where insertion or deletion of a few amino acids was introduced, is shown as an inverted triangle. (B) The structure of the latent form (Mottonen et al. 1992; 1DVN.pdb). The β-sheets A, the RCL, and the first strand of β-sheet C (s1C) are colored as in the native form. The N-terminal portion of the scissile peptide bond is inserted into β-sheet A without cleavage, forming the fourth strand of the β-sheet A (s4A; indicated in light gray). These figures were prepared using ViewerLite (Ac-celrys Inc.).

Figure 2.

Conformational conversion of PAI-1 mutants. The wild-type (Wt) and mutant PAI-1 proteins were incubated in a buffer (45 mM phosphate, 70 mM NaCl, 0.01% Tween 80, pH 7.4) at 37°C for 1 h. Conformational conversion was monitored on transverse urea gradient gels, containing a gradient of 0~8 M urea perpendicular to the direction of electrophoresis. The migration positions of the native and the urea-stable forms are indicated with arrows.

Figure 3.

Conformational stability of native PAI-1 variants. Urea-induced equilibrium unfolding transitions were measured by changes in fluorescence emission intensity at 333 nm (λ_ex = 295 nm). (•) The native wild-type; (▾) the native Ins1; (▪) the native Ins2; (○) the native Del1; (▿) the native Del2.

Figure 4.

Limited proteolysis of PAI-1 to probe the conformation of the RCL in the urea-stable form. The native wild-type (Wt), native Ins1, native Del1, and the latent wild-type, Ins1, and Ins2 proteins were treated with trypsin (T), porcine pancreatic elastase (PE), Staphylococcus aureus V8 protease (V8), or tPA. Digestion patterns were analyzed by 12% SDS-polyacrylamide gel electrophoresis. The migration positions of molecular mass standards (lane M; Bio-Rad Co., low range) are indicated on the left of the gels. U is the untreated PAI-1 protein. The locations of inhibitory complexes with tPA (Cp), tPA, un-reacted PAI-1 (PAI-1), the RCL-cleaved PAI-1 (Cl_RCL), and two cleaved latent PAI-1 fragments by trypsin (Cl_T) are marked accordingly.

Figure 5.

Conformational conversion rates of PAI-1 variant proteins. (A) Conversion rates were followed by appearance of the urea-stable form on polyacrylamide gels containing 4 M urea. The migration positions of the native form and the latent form are indicated with arrow heads. (B) Inhibitory complex formation of the native PAI-1 variants with tPA. One microgram of native PAI-1 proteins was incubated with (+) or without (−) 3 μg of tPA, and the formation of the SDS-resistant PAI-1-tPA complex was analyzed by 10% SDS-polyacrylamide gel electrophoresis. The locations of inhibitory complexes (Cp), tPA, un-reacted PAI-1 (PAI-1) and cleaved PAI-1 (Cl) are marked accordingly. (Lane M) Molecular mass standards (Bio-Rad Co., low range). (C) The conversion rates of the wild-type, Ins1, and Del1 proteins were followed by inhibitory activity loss. After PAI-1 proteins were incubated at 37°C for various times, the remaining inhibitory activity was measured with urokinase using Spectrozyme UK as a substrate. (•) The wild-type; (▾) Ins1; (○) Del1 PAI-1.