Tài liệu Báo cáo Y học: Importance of the amino-acid composition of the shutter region of

Importance of the amino-acid composition of the shutter region of

plasminogen activator inhibitor-1 for its transitions to latent and

substrate forms

Martin Hansen, Marta N. Busse and Peter A. Andreasen

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

The serpins are of general protein chemical interest due to

their ability to undergo a large conformational change

consisting of the insertion of the reactive centre loop (RCL)

as strand 4 of the central b sheet A. To make space for the

incoming RCL, the ‘shutter region’ opens by the b strands

3A and 5A sliding apart over the underlying a helix B. Loop

insertion occurs during the formation of complexes of

serpins with their target serine proteinases and during

latency transition. This type of loop insertion is unique to

plasminogen activator inhibitor-1 (PAI-1). We report here

that amino-acid substitutions in a buried cluster of three

residues forming a hydrogen bonding network in the shutter

region drastically accelerate a PAI-1 latency transition; that

the rate was in all cases normalized by the PAI-1 binding

protein vitronectin; and that substitution of an adjacent b

strand 5A Lys residue, believed to anchor b strand 5A to

other secondary structural elements, had differential effects

on the rates of latency transition in the absence and the

presence of vitronectin, respectively. An overlapping, but

not identical set of substitutions resulted in an increased

tendency to substrate behaviour of PAI-1 at reaction with its

target proteinases. These findings show that vitronectin

regulates the movements of the RCL through conformation￾al changes of the shutter region and b strand 5A, are in

agreement with RCL insertion proceeding by different

routes during latency transition and complex formation, and

contribute to the biochemical basis for the potential use of

PAI-1 as a therapeutic target in cancer and cardiovascular

diseases.

Keywords: cancer; extracellular proteolysis; fibrinolysis;

proteinase inhibitors; serine proteinases.

The serpins constitute a protein family of which the best

characterized members are serine proteinase inhibitors,

including antithrombin III, a1-antitrypsin, and plasminogen

activator inhibitor-1 (PAI-1). The serpins are globular

proteins consisting of nine a helices and three b sheets

(reviewed in [1–3]). Serpins are of general protein chemical

interest due to their ability to undergo a large confor￾mational change with the insertion of the surface-exposed

reactive centre loop (RCL) as strand 4 of the large central b

sheet A as the main event (Fig. 1). The RCL insertion

results in a considerable stabilization compared to the native

serpin structure, and is often referred to as the stressed-to￾relaxed transition (for a review, see [2]). This stabilization

forms the basis for the mechanism behind the inhibitory

function of serpins. After cleavage of the P1 –P1

0 peptide

bond in the RCL, the active site serine of the proteinase

remains attached to the carboxyl group of the P1 residue by

an ester bond [4–6]. The subsequent RCL insertion into

b sheet A therefore results in an < 7-nm translocation of

the proteinase from the position of its initial encounter with

the RCL to the other pole of the serpin [7–10]. The

translocation results in distortion of the proteinase [11] and

inactivation of the enzymatic machinery [10]. Delayed RCL

insertion results in hydrolysis of the ester bond, the serpin

thus behaving as an ordinary substrate [12]. The stabil￾ization caused by RCL insertion also underlies the unique

conversion of active PAI-1 to the latent state, in which the

N-terminal part of the intact RCL is inserted as b strand 4A

without cleavage of any peptide bonds, and the C-terminal

part is stretched along the surface of the molecule [13]

(Fig. 1).

In order to make space for the incoming new strand

during RCL insertion, a fragment of the structure consisting

of b strands 1A, 2A, 3A, and a helix F (the small serpin

fragment) must slide away from the rest of the structure (the

large serpin fragment). During the b sheet opening, the

region around a helices D and E forms a flexible joint, and

b strands 3A and 5A slide apart in a shutter-like manner over

the underlying a helix B [14]. The central part of b strands

3A and 5A and the N-terminal part of a helix B is therefore

referred to as the shutter region [2]. By high resolution X-ray

crystal structure analysis of the native form of the serpin

plasminogen activator inhibitor-2 (PAI-2) and the P1 –P1

0

cleaved form of horse leukocyte elastase inhibitor, a buried

Enzymes: Urokinase-type plasminogen activator (EC 3.4.21.73).

Note: plasminogen activator inhibitor-1 and vitronectin have the NCBI

accession numbers P05121 and P04004, respectively.

Note: a website is available at http://www.mbio.aau.dk

Correspondence to M. Hansen, Laboratory of Cellular Protein Science,

Department of Molecular and Structural Biology, University of Aarhus,

10C Gustav Wieds Vej, 8000 Aarhus C, Denmark.

Fax: þ 45 86123178, Tel.: þ 45 89425079,

E-mail: [email protected]

(Received 16 July 2001, revised 5 October 2001, accepted

8 October 2001)

Abbreviations: HEK293T, the human embryonic kidney cell line 293T;

LMW-uPA, low Mr uPA; PAI-1, plasminogen activator inhibitor-1;

PAI-2, plasminogen activator inhibitor-2; RCL, reactive centre loop;

S-2444, L-5-pyroglutamyl-glycyl-L-arginine-p-nitroaniline; uPA,

urokinase-type plasminogen activator.

Eur. J. Biochem. 268, 6274–6283 (2001) q FEBS 2001