Proximity of the catalytic region and the kringle 2 domain in the closed conformer of plasminogen

224

Biochimica et Biophy.sica Acta 832 (1985) 224-227

Elsevier BBA 30114

BBA Report

P r o x i m i t y of the catalytic region and the kringle 2 d o m a i n in the closed c o n f o r m e r of p l a s m i n o g e n Lfiszl6 B f i n y a i a n d Lfiszl6 P a t t h y Institute of Enzymologv, Biological Research Center. Hungarian A cademy of Sciences, H-1502 Budapest (Hungao')

(Received June 12th, 1985) Revised manuscript received September20th, 1985)

Key words: Plasminogen; Urokinase; Kringle 2 domain: Chemical cross-linking; (Human)

Introduction of a single intramolecular cross-link with 1,5-difluoro-2,4-dinitrobenzene into Glu-plasminogen freezes the molecule in its closed conformational state (B/royal, L. and Patthy, L. (1984) J. Biol. Chem. 259, 6466-6471). Here we show that the cross-link connects Lys-203 of the kringle 2 domain and Tyr-671 of the catalytic domain, indicating that these regions are in close proximity in the closed conformer of Giu-plasminogen. Comparison of the parameters of the urokinase-catalysed activation of native and cross-linked Glu-plasminogen species indicates that cross-linking of kringle 2 and the catalytic region interferes with the productive binding of u(okinase to plasminogen.

Glu-plasminogen contains a large non-proteinase region linked to the amino-terminal end of the trypsin-homologue catalytic region [l]. This non-proteinase part interferes with the activation of plasminogen, as indicated by the observation that removal of this region, i.e., conversion of plasminogen to miniplasminigen (Va1442-plasminogen), increases the catalytic efficiency of urokinase-catalysed plasminogen activation [2]. The second-order rate constant of plasminogen activation with urokinase is also increased by lysine analogues [2-7] or by conversion of Glu-plasminogen to Lys-plasminogen [2,3,6,7], concomitant with transition of plasminogen from a compact, closed conformation to a looser structure [3-5]. These observations suggest that in the closed conformational state of native Glu-plasminogen the non-proteinase part somehow hinders the urokinase-catalysed activation. In our previous studies we have shown that 1,5-difluoro-2,4-dinitrobenzene treatment of Gluplasminogen specifically cross-links the kringle 1 + 2 + 3 segment of the non-proteinase part to the

catalytic region via an N',O-2,4-dinitrophenylenetyrosyl-lysyl cross-link and this cross-link freezes the molecule in a conformational state which is not favourable for activation [8]. In the present study we show that Lys-203 of the kringle 2 domain and Tyr-671 of the catalytic region are the residues which are cross-linked and suggest that these two regions must be allowed to separate in order to permit more rapid plasminogen activation with urokinase. Human native plasminogen, Glu-plasminogen, was prepared from fresh citrated plasma by affinity chromatography on lysine-Sepharose 4B [9] in the presence of bovine pancreatic trypsin inhibitor (Trasylol, Bayer). The concentration of plasminogen solutions was determined using A2~ 1,~0 = 16.8 [10]. Glu-plasminogen was cross-linked with 1,5difluoro-2,4-dinitrobenzene (Serva), the modified molecule was digested with porcine pancreatic elastase (Serva) and the fragments were separated by gel filtration on a Sephadex G-75 column equilibrated with 0.3 M ammonium bicarbonate as described previously [8].

0167-4838/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

225

The 70 kDa elastase fragment (kringle 1 + 2 + 3 + miniplasminogen, Ref. 8) which contains the cross-link was reduced by incubating the protein (10 mg/ml) in 0.1 M Tris-HCl/5 mM EDTA/6 M guanidine hydrochloride (pH 8.0) buffer with 5% (v/v) 2-mercaptoethanol at 25°C for 30 min. The reduced protein was alkylated for 30 min with iodoacetic acid used in 2-fold molar excess over thiol-groups, the sample was desalted by gel filtration on a Sephadex G-25 column in 0.1 M ammonium bicarbonate and the protein was lyophilized. The S-carboxymethylated protein was digested for 3 h with L-l-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Worthington) in 0.1 M ammonium bicarbonate (pH 8.0) at 37°C at an enzyme-to-substrate ratio of 1 : 50, the digestion was arrested with diisopropyl phosphofluoridate (Merck) and the sample was lyophilized. The tryptic digest was subjected to hydrolysis with chymotrypsin (Worthington) for 3 h in 0.1 M ammonium bicarbonate, pH 8.0, at 37°C at an enzyme-to-substrate ratio of 1:50. The trypticchymotryptic peptide (peptide TC) containing the cross-link was isolated by high voltage paper electrophoresis at pH 6.5 and pH 1.9 and paper chromatography in 1-butanol/pyridine/water (45 : 45 : 60) using Whatman No. 3MM papers; the labeled peptide was detected by its yellow color (Table I). Sequential Edman degradation was performed according to the method of Gray and Hartley [11] and the phenylthiohydantoin- and dansyl-derivatives of the aminoterminal amino acids were identified according to described methods [12,13]. Sequence analysis showed that the cross-linked peptide contained isoleucine in the first position and proline plus valine in the second position (Fig. 1). The cross-linked peptide (10 mg/ml) was subjected to digestion with thermolysin (Calbiochem, 0.4 mg/ml) in 0.1 M ammonium bicarbonate (pH 8.0) at 37°C for 6 h and the two resulting peptides were isolated by pH 1.9 high-voltage paper electrophoresis (Table I). The amino acid sequence of peptide TC-Th 1, determined by the dansyl-Edman method [11,12], Val-Ala-Asp-Arg, showed that it corresponds to residues 673-676 of plasminogen [1]. This peptide was formed by cleavage of peptide T C at the Va1672-Va1673 bond, the tyrosine residue of the N',O-2,4-dinitrophenylene-tyrosyl-lysyl cross-link

TABLE I AMINO ACID COMPOSITIONS AND ELECTROPHORETIC MOBIL1TIES OF THE PEPTIDES CONTAINING THE N~,O-2,4-DINITROPHENYLENE-TYROSYL EYSYL CROSS-LINK The peptides were hydrolyzed in 6 M HCI at II0°C for 24 h and the composition of the acid hydrolysates was determined by amino acid analysis on a Biotronik LG 2000 analyzer. Peptides Tc-Th 1 and TC-Th 2 were derived from trypticchymotryptic peptide (TC) by thermolytic digestion. Numbers in parenthesis are residue values based on sequence [1]. Electrophoretic mobilities are expressed relative to alanine and arginine at pH 1.9 and pH 6.5. respectively, N',O-2,4-dinitrophenylene-tyrosyl-lysine was detected on pH 1.9 paper electropherograms of acid hydrolysates. Residue

Aspartic acid Serine Proline Alanine Valine Isoleucine Pbenylalanine Lysine Arginine N', O-2,4-dinitrophenylene-tyrosyllysine Mobility at pH 1.9 at pH 6.5

Peptide TC

TC-Th 1

TC-Th 2

1.6 (2) 0.9 (1) 2.0 (2) 1.0 (1) 1.6 (2) 0.8 (1) 1.0 (1) 1.1 (1) 1.0 (1)

0.8 (1)

0.9 (1) 1.1 (1) 1.9 (2)

+ 0.90 0.09

1.0 (1) 0.9 (1) 0.9 (1) 1.0 (1) 1.2 (1) 1.0 (1)

1.00 0.09

+ 0.78 0.08

corresponds to Tyr-671 of the catalytic chain of plasminogen (Fig. 1). Sequence analysis of peptide TC-Th 2 gave the unique sequence, Ile-Pro-Ser-XPhe which corresponds to residues 200-204 of the kringe 2 domain [1], the lysine residue in the fourth position of this peptide, Lys-203, is involved in cross-linking with Tyr-671 (Fig. 1). The results of structural studies thus indicate that the crosslink was formed between the kringle 2 domain and the catalytic region. Kinetic analysis of the activation of native and cross-linked Glu-plasminogen with urokinase was performed essentially as described previously [8]. A two-stage plasminogen activation assay similar to that used by Hoylaerts et al. [14] was employed. The activation mixture contained the plasminogen derivative (5-200 /~M) and urokinase (Leo Pharmaceuticals, 2-9 IU/ml) in 50 mM Tris-HC1/150

226

(a)

1200

]

-A-C-L-P-S-P-N-~Y-V-V-A-D-R'~T-E-C-

67o I

!

TABLE II

210 F-

680

(b) i _~p.~S~-K _ F~--p - N~KK Y (c) V-A-D-R

Fig. 1. Localization of the N ~ , O - 2 , 4 - d i n i t r o p h e n y l e n e - t y r o s y l lysyl cross-link in plasminogen. (a) The enboxed residues correspond to the tryptic-chymotryptic peptide (TC); the solid bar represents the cross-link. The numbers indicate the position of the residues in the sequence of Glu-plasminogen [1]. (b) and (,~) Peptides obtained by thermolysin digestion of peptide TC the residues marked with ( ---, ) were identified by the dansyl-Edman method [11,12]. The standard IUPAC one-lener code for amino acid residues is used.

mM NaCI (pH 7.4) buffer. The solution was incubated at 37°C for 2 min, aliquots were diluted 20-fold into assay buffer preincubated at 25°C and the activity of plasmin (or plasmin analogue) formed was determined spectrophotometrically with DVal-Leu-Lys-pNA (S-2251, KABI) as described previously [8]. The assay mixture containing plasmin and the substrate (0.6 mM) in 50 mM Tris-HCl/150 mM NaCI (pH 7.4) buffer was incubated at 25°C in the thermostatically controlled cell holder of a Varian 634 spectrophotometer and the rate of hydrolysis of substrate was registered at 405 nm. The short activation time ensured that during the activation only a small amount of plasmin was formed and this did not lead to the formation of Lys-plasminogen as revealed by gel electrophoretic analysis of the samples according to the method of Walther et al. [15]. The kinetic parameters of urokinase-catalysed activation of native and cross-linked Glu-plasminogen in the presence and absence of ~-aminocaproic acid are summarised in Table II. The results indicate that both native and cross-linked Glu-plasminogen have weak affinity for urokinase. In the presence of c-aminocaproic acid the K,n of native Glu-plasminogen for urokinase decreases (as also noted in Refs. 6, 7), but no significant change in binding parameter is observed in the case of cross-linked Glu-plasminogen. The results

KINETIC CONSTANTS FOR THE ACTIVATION OF GluP L A S M I N O G E N A N D C R O S S - L I N K E D Glu-PLASMINOGEN WITH UROKINASE Plasminogen species

Glu-plasminogen Glu-plasminogenwith c-aminocaproicacid (5 mM) Cross-linked Gluplasminogen Cross-linked Gluplasminogen with ~-aminocaproicacid (5 mM)

k~,~t

K m

kc,,t/K,1

(s 1)

(~M)

(~ is 7)

3.8

138

0.028

3.8

18

0.211

1.1

215

0.005

0.8

238

0.003

indicate that the conformational change induced by ligand is a prerequisite of the more efficient binding of urokinase. Since this conformational change is prevented by the cross-link [8] introduced between the kringle 2 domain and the catalytic chain this may be the reason why the activation parameters of cross-linked plasminogen are insensitive to c-aminocaproic acid. Nevertheless, the possibility can not be ruled out that the crosslink changed some other steric or charge properties of the protein and these changes are responsible for the impairment of plasminogen activation. Electron microscopic studies on native Gluplasminogen have shown the molecule to be coiled into a spiral in a way that the two termini of the 20-24 n m × 2.2-2.5 nm filament-like molecule are brought into juxtaposition [16]. It seems tempting to assume that in this molecular model the point of the juxtaposition corresponds to the areas of the kringle 2 domain and the catalytic domain that are close to each other in the closed conformer. However, our results do not permit any conclusion as to whether these areas have a direct role in maintaining the closed conformation or that they are brought in proximity by other intramolecular interactions. Similarly, it cannot be decided at present whether the cross-linked surfaces are involved in urokinase binding or that the geometry of the spiral is such that it hinders the proper alignment of urokinase and Glu-plasminogen.

227

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