Tài liệu Báo cáo khoa học: Mutational analysis of plasminogen activator inhibitor-1 Interactions of

Mutational analysis of plasminogen activator inhibitor-1

Interactions of a-helix F and its neighbouring structural elements regulates

the activity and the rate of latency transition

Troels Wind*, Jan K. Jensen, Daniel M. Dupont, Paulina Kulig and Peter A. Andreasen

Laboratory of Cellular Protein Science, Department of Molecular Biology, Aarhus University, Denmark

The serpin plasminogen activator inhibitor-1 (PAI-1) is a

fast and specific inhibitor of the plasminogen activating

serine proteases tissue-type and urokinase-type plasminogen

activator and, as such, an important regulator in turnover of

extracellular matrix and in fibrinolysis. PAI-1 spontaneously

loses its antiproteolytic activity by inserting its reactive centre

loop (RCL) as strand 4 in b-sheet A, thereby converting to

the so-called latent state. We have investigated the import￾ance of the amino acid sequence of a-helixF (hF) and the

connecting loop to s3A (hF/s3A-loop) for the rate of latency

transition. We grafted regions of the hF/s3A-loop from

antithrombin III and a1-protease inhibitor onto PAI-1,

creating eight variants, and found that one of these rever￾sions towards the serpin consensus decreased the rate of

latency transition. We prepared 28 PAI-1 variants with

individual residues in hF and b-sheet A replaced by an

alanine. We found that mutating serpin consensus residues

always had functional consequences whereas mutating

nonconserved residues only had so in one case. Two variants

had low but stable inhibitory activity and a pronounced

tendency towards substrate behaviour, suggesting that

insertion of the RCL is held back during latency transition as

well as during complexformation with target proteases. The

data presented identify new determinants of PAI-1 latency

transition and provide general insight into the characteristic

loop–sheet interactions in serpins.

Keywords: alignment; conformation; mutational analysis;

PAI-1; proteases; serpin.

Plasminogen activator inhibitor-1 (PAI-1) is the primary

inhibitor of both urokinase-type and tissue-type plasmino￾gen activator (uPA and tPA, respectively) and as such is an

important regulator of physiological events in which

plasmin-catalysed extracellular proteolysis is involved.

PAI-1 belongs to the serine protease inhibitor (serpin)

family whose antiproteolytic activity is governed by their

structural flexibility. In the active serpin conformation, the

reactive centre loop (RCL) with the P1–P1¢ bait peptide

bond is surface exposed. Formation of the covalent serpin–

protease complexinvolves a Michaelis docking complex,

cleavage of the P1–P1¢ peptide bond, linkage of the active

site Ser of the protease to the carboxyl group of P1 by an

ester bond and insertion of the N-terminal part of the RCL

as strand 4 in b-sheet A (s4A) of the serpin. Consequently,

the protease is trapped in a covalent acyl-enzyme complexin

which its reactive site is distorted, as illustrated by the crystal

structure of the complexbetween a1-protease inhibitor

(a1PI, also referred to as a1-antitrypsin) and trypsin [1].

Under some conditions, however, RCL insertion is delayed,

resulting in hydrolysis of the ester bond, release of free

protease and insertion of the cleaved RCL as s4A. This

pathway is referred to as substrate behaviour of the serpin.

Complexformation between serpins and their cognate

proteases is fuelled by the thermodynamic properties of the

serpin. Accordingly, insertion of the RCL as s4A and

the ensuing structural rearrangements of the serpin stabilizes

the molecule in a so-called
relaxed conformation, as

opposed to the metastable
stressed conformation with the

RCL exposed on the surface (reviewed in [2–4]).

PAI-1 spontaneously converts into a relaxed conforma￾tion at a significant rate without cleavage of the RCL (for a

review see [5]). During this structural transformation,

referred to as latency transition, the N-terminal part of

the intact RCL is inserted as s4A [6] (Fig. 1). Latent versions

of the serpins antithrombin III (ATIII) [7], a1-protease

inhibitor (a1PI) [8], and a1-antichymotrypsin (a1ACT) [9]

have also been isolated, but none of these undergo this

transition as readily as PAI-1. The physiological role of

PAI-1 latency transition, if any, remains elusive [5].

Some PAI-1 variants with single mutations and modest

decreases in the rate of latency transition have been

obtained through heuristic protein engineering [10,11] while

others have been identified by chance [12–15]. The variants

with the slowest latency transition carry multiple mutations

Correspondence to J. K. Jensen, Laboratory of Cellular Protein

Science, Department of Molecular Biology, Aarhus University,

Gustav Wieds Vej 10C, 8000 A˚rhus C, Denmark.

Fax: + 45 86123178, Tel.: + 45 89425074,

E-mail: [email protected]

Abbreviations: PAI-1, plasminogen activator inhibitor-1; RCL,

reactive centre loop; a1PI, a1-protease inhibitor (a1-antitrypsin);

a1ACT, a1-antichymotrypsin; ATIII, antithrombin III;

hF, a-helixF; HMK, heart muscle kinase.

Enzyme: uPA, urokinase-type plasminogen activator (EC 3.4.21.73).

*Present address: Centre for Vascular Research, School of Medical

Sciences, The University of New South Wales,

Sydney NSW 2052, Australia.

(Received 4 December 2002, revised 7 February 2003,

accepted 13 February 2003)

Eur. J. Biochem. 270, 1680–1688 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03524.x