Solvent Effects on Activity and Conformation of Plasminogen Activator Inhibitor-1
P. A. Andreasen
1
From the Department of Molecular and Structural Biology, Aarhus University, Denmark
,
R. Egelund
1
From the Department of Molecular and Structural Biology, Aarhus University, Denmark
,
S. Jensen
1
From the Department of Molecular and Structural Biology, Aarhus University, Denmark
,
K. W. Rodenburg
1
From the Department of Molecular and Structural Biology, Aarhus University, Denmark
› Author AffiliationsThis work was supported financially by the Danish Cancer Society, the Danish Heart Foundation, the Danish Medical Research Council, the Danish Biotechnology Programme, Aarhus University Research Foundation, and the NOVO-Nordisk Foundation.
We have studied effects of the solvent composition on the activity and the conformation of human plasminogen activator inhibitor-1 (PAI-1) from HT-1080 fibrosarcoma cells. Non-ionic detergents, including Triton X-100, reduced the inhibitory activity of PAI-1 more than 20-fold at 0° C, but less than 2-fold at 37° C, while glycerol partly prevented the detergent-induced activity-loss at 0° C. The activity-loss was associated with an increase in PAI-1 substrate behaviour. Evaluating the PAI-1 conformation by proteolytic susceptibility of specific peptide bonds, we found that the V8-proteinase susceptibility of the Glu332-Ser333 (P17-P16) bond, part of the hinge between the reactive centre loop (RCL) and β-strand 5A, and the endoproteinase Asp-N susceptibility of several bonds in the β-strand 2A-α-helix E region were increased by detergents at both 0 and 37° C. The susceptibility of the Gln321-Ala322 and the Lys325-Val326 bonds in β-strand 5A to papain and trypsin, respectively, was increased by detergents at 0° C, but not at 37° C, showing a strict correlation between proteinase susceptibility of β-strand 5A and activity-loss at 0° C. Since the β-strand 2A-α-helix E region also showed differential susceptibility to endoproteinase Asp-N in latent, active, and reactive centre-cleaved PAI-1, we propose that a detergent-induced conformational change of the β-strand 2A-α-helix E region influences the movements of β-sheet A, resulting in a cold-induced conformational change of β-strand 5A and thereby an increased substrate behaviour at low temperatures. These results provide new information about the structural basis for serpin substrate behaviour.
References
1
Potempa J,
Korzus E,
Travis J.
The serpin superfamily of proteinase inhibitors: structure, function, and regulation. J Biol Chem 1994; 269: 15957-60.
3
Andreasen PA,
Kjøller L,
Christensen L,
Duffy MJ.
The urokinase-type plasminogen activator system in cancer metastasis. A review. Int J Cancer 1997; 72: 1-22.
5
Huber R,
Carrell RW.
Implications of the three-dimensional structure of α1-antitrypsin for structure and functions of serpins. Biochemistry 1989; 28: 8951-66.
7
Lawrence DA,
Ginsburg D,
Day DE,
Berkenpas MB,
Verhamme IM,
Kvassman JO,
Shore JD.
Serpin-protease complexes are trapped as stable acyl-enzyme intermediates. J Biol Chem 1995; 270: 25309-12.
8
Wilczynska M,
Fa M,
Ohlsson I P,
Ny T.
The inhibition mechanism of ser-pins. Evidence that the mobile reactive center loop is cleaved in the native protease-inhibitor complex. J Biol Chem 1995; 270: 29652-5.
9
Egelund R,
Rodenburg KW,
Andreasen PA,
Rasmussen MS,
Guldberg RE,
Petersen TE.
An ester bond linking a fragment of a serine proteinase to its serpin inhibitor. Biochemistry 1998; 37: 6375-9.
11
Wilczynska M,
Fa M,
Karolin J,
Ohlsson PI,
Johansson LBÅ,
Ny T.
Struc tural insights into serpin-protease complexes reveal the inhibitory mechanism of serpins. Nat Struct Biol 1997; 4: 354-7.
14
Plotnick I M,
Mayne L,
Schechter NM,
Rubin H.
Distortion of the active site of chymotrypsin complexed with a serpin. Biochemistry 1996; 35: 7586-90.
15
Kaslik G,
Patthy A,
Bálint M,
László G.
Trypsin complexed with α1-proteinase inhibitor has an increased structural flexibility. FEBS Lett 1995; 370: 179-83.
16
Kaslik G,
Kardos J,
Szabo E,
Szilagyi L,
Zavodszky P,
Westler WM,
Markley JL,
Graf L.
Effects of serpin binding on the target proteinase: global stabilization, localized increased structural flexibility, and conserved hydrogen bonding at the active site. Biochemistry 1997; 36: 5455-64.
17
Stavridi ES,
O´Malley K,
Lukacs CM,
Moore WT,
Lambris JD,
Christianson DW,
Rubin H,
Cooperman BS.
Structural change in α-chymotrypsin induced by complexation with α1-antichymotrypsin as seen by enhanced sensitivity to proteolysis. Biochemistry 1996; 35: 10608-15.
18
Gils A,
Declerck PJ.
Modulation of plasminogen activator inhibitor 1 by Triton X-100. Identification of two consecutive conformational transitions. Thromb Haemost 1998; 80: 286-91.
21
Kjøller L,
Kanse SM,
Kirkegaard T,
Rodenburg KW,
Rønne E,
Goodmann SL,
Preissner KT,
Ossowski L,
Andreasen PA.
Plasminogen activator inhibitor-1 represses integrin- and vitronectin-mediated cell migration independently of its function as an inhibitor of plasminogen activation. Exp Cell Res 1997; 232: 420-9.
22
Andreasen PA,
Riccio A,
Welinder KG,
Douglas R,
Sartorio R,
Nielsen LS,
Oppenheimer C,
Blasi F,
Danø K.
Plasminogen activator inhibitor type 1: reactive center and amino-terminal heterogeneity, determined by protein and cDNA sequencing. FEBS Lett 1986; 209: 213-8.
23
Grøndahl-Hansen J,
Nielsen LS,
Kristensen P,
Grøndahl-Hansen V,
Andreasen PA,
Danø K.
Plasminogen activator in psoriatic scales is of the tissue-type PA as identified by monoclonal antibodies. Br J Dermatol 1985; 113: 257-63.
26
Gekko K,
Timasheff SN.
Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures. Biochemistry 1981; 20: 4667-76.
28
Hubbard SJ,
Eisenmenger F,
Thornton JM.
Modeling studies of the change in conformation required for cleavage of limited proteolytic sites. Protein Science 1994; 3: 757-68.
29
Lawrence DA,
Berkenpas MB,
Palaniappan S,
Ginsburg D.
Localization of vitronectin binding domain in plasminogen activator inhibitor-1. J Biol Chem 1994; 269: 15223-8.
