Studies on the chemical nature of lysine-binding sites and on their localization in human plasminogen

374 Biochimica et Biophysiea Acta, 625 (1980) 374--378

© Elsevier/North-Holland Biomedical Press

BBA Report BBA 31314 STUDIES ON THE CHEMICAL N A T U R E OF LYSINE-BINDING SITES AND ON T H E I R LOCALIZATION IN HUMAN PLASMINOGEN

PETER G. LERCH* and EGON E. RICKLI** Institute of Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne (Switzerland)

(Received March 10th, 1980) Key words: L ysine-binding site; Plasminogen ; 'Kringle '; (Human)

Summary The isolated 'kringie' structures 1 and 4 of human plasminogen lost lysine affinity upon photo-oxidation of histidine, b u t mostly retained it in the presence of 6-aminohexanoic acid. Lysine affinity was lost and could be partially restored after blocking of histidine with diethylpyrocarbonate and deblocking, or after esterification of COOH-groups and saponification. Only His-31 and most likely Asp-54 qualify as participants in a lysine binding site when the primary structures of the 'kringles' are considered.

Plasminogen contains several sites, one of high and approximately four of low affinity [1--3] which are responsible for complex formation with co-aminocarboxylic acids (6-aminohexanoic acid, t r a n s - 4 - a m i n o m e t h y l - c y c l o hexane carboxylic acid, lysine, etc.). These sites (generally referred to as lysine-binding sites) are located in that part of the plasminogen polypeptide chain which after activation becomes the heavy (or A - ) chain of plasmin [4] After limited digestion of plasminogen with elastase Sottrup-Jensen et al. [5] demonstrated that of the five heavy chain 'kringle' structures the isolated 'kringle' 4 and a fragment consisting of 'kringles' 1+2+3 could bind lysine whereas 'kringle' 5 could not. Later, we showed lysine binding with isolated 'kringle' 1 [6]. According to a model of Wiman and Collen [7] the lysinebinding sites may play a crucial role in the regulation of physiological fibrinolysis. In this communication we report the results of chemical modification of amino acid residues in the isolated 'kringles' 1 and 4, aimed at the identification and localization of lysine-binding sites in the primary structure of the ' kringles'. *Present address: T h e o d o r K o e h e r I n s t i t u t e , Freiestrasse 1, C H - 3 0 0 0 B e r n e 9, S w i t z e r l a n d . **To whom correspondence should be addressed.

375 The isolation of human plasminogen was carried out as described by Rickli and Cuendet [8]. For the preparation of the fragments comprising 'kringles' 1+2+3 and 'kringle' 4 by partial digestion of plasminogen with porcine pancreatic elastase a modification [6] of the m e t h o d of SottrupJensen et al. [5] was used. 'Kringle' 1 was isolated after c h y m o t r y p t i c degradation of 'kringles' 1+2+3 as described by Lerch et al. [6]. Affinity chromatograpl~ic tests of chemically modified 'kringle' polypeptides were carried out on an affinity column of lysine-Biogel (2 X 5 cm), equilibrated against 50 mM sodium phosphate buffer, pH 7.4. 0.2--2 mg polypeptide, dissolved in the same buffer, were applied, the column was washed free of unadsorbed material, followed by the elution of specifically adsorbed material with the same buffer containing 10 mM 6-aminohexanoic acid (flow rate approx. 100 ml/h). The absorbance of the eluate was recorded at 280 nm with a ultraviolet monitor. The amount of adsorbed material expressed as a percentage of the total amount of polypeptide applied to the column was taken as a measure of the lysine-binding capacity remaining after chemical modification. The peptide cleavage with CNBr, reduction of disulfide bonds and alkylation with iodoacetamide was carried out as reported previously [9]. For the photo-oxidation of histidine residues [10] peptide was dissolved in 0.1 M sodium phosphate buffer, pH 6.5 (1 mg/ml) and the solution was made 0.01% in methylene blue. Experiments were performed in the presence (10 mM) as well as in the absence of 6-aminohexanoic acid. Samples were illuminated for various lengths of time (1--60 min) at 30°C b y two 150 W tungsten lamps, placed at a distance of 5 cm on opposite sides of the sample tube. The peptide material was subsequently purified in the dark by gel filtration on a Sephadex G-50 (sf) column (2.5 X 40 cm) in 0.1 M NH4HCO3 and lyophilized. In addition, histidine residues were modified reversibly with diethylpyrocarbonate according to Miles and Kumagi [11]. Deblocking t o o k place by incubating in hydroxylamine for 12 h. For arginine residues the modification m e t h o d of Patthy and Smith [12] with cyclohexanedione was used. Maleic anhydride was used for the reversible blocking of lysine residues as described by Butler et al. [13]. The following conditions were used for the esterification of carboxyl groups: peptide material, suspended in anhydrous methanol containing 0.1 M HC1 was stirred for 24 h at room temperature, dialyzed for 2 h against 10 -3 M HC1 at 4°C and lyophilized [14]. Saponification of the esters formed was carried out by incubation in 0.2 M NH4HCO3 for 24 h at 20°C. Iodination of tyrosine residues was performed according to Azari and Feeney [15]. Peptide material (0.6 mg) was dissolved in 200 pl 0.1 M borate buffer, pH 9.5. Then two 10 ~l portions of a solution of 50 mM I2 and 0.24 M KI were added at 0°C. After 15 min the reaction mixture was purified by gel filtration as described above. Reduction and alkylation of the intra-'kringle' disulfide bridges caused a complete loss of the binding capacity of 'kringles' 1 and 4 for lysineBiogel.

376

a)

Sf ~¢

c

M

b) 1C-C

Fig. 1. S c h e m a t i c r e p r e s e n t a t i o n o f the " k r i n g l e s " 1 (a) a n d 4 (b) a c c o r d i n g to S o t t r u p - J e n s e n et al. [ 5 ] . N u m b e r i n g o f a m i n o acid residues s t a r t s at t h e first h a l f - c y s t i n e residue. A r r o w s i n d i c a t e posit i o n s of CNBr cleavage. H i s ( 3 1 ) a n d A s p ( 5 4 ) are s u p p o s e d t o be i m p l i c a t e d in b i n d i n g o f e - a m i n o c a r b o x y l i c acids.

When 'kringle' 4 was treated with CNBr in 70% formic acid only the methionine-asparagine bond (positions 47-48) was cleaved [6] (the numbering of residues starts at the first half-Cys residue of the 'kringle', see also Fig. 1). This resulted in a complete loss of the binding capacity for lysineBiogel, whereas incubation for the same time in 70% formic acid alone did not affect this property. The first 'kringle', however, retained its full lysine binding capacity, although CNBr cleaved the methionine-serine bond (positions 13-14). Photo-oxidation of histidine significantly influenced the lysine-binding capacity of both 'kringles' 1 and 4. In the absence of 6-aminohexanoic acid they lost 90% of their binding capacity after 5 min of photo-oxidation. 6-Aminohexanoic acid added to the oxidation medium exerted a protective influence, since after 5 min of oxidation only a 20% loss of the binding capacity was observed and after 1 h of treatment 50% of the polypeptide still adsorbed to lysine-Biogel. Thus in the complex with 6-aminohexanoic acid the destruction of the binding ability is considerably retarded. Amino acid analyses of photooxidized 'kringles' showed a greater loss of histidine in the non-adsorbing portion than in the peptide fraction adsorbed to lysine-Biogel with a difference approaching one residue. Noticeable decreases in the content of other amino acids were not observed. The treatment of 'kringle' 4 with diethylpyrocarbonate also resulted in a loss of the binding capacity. After deblocking with hydroxylamine the binding properties were restored. After treatment with cyclohexanedione the binding capacity remained unchanged. The blocking of lysine residues abolished completely the binding capacity of 'kringle' 4, whereas that of 'kringle' 1 was hardly affected. Deblocking of maleylated lysine residues restored the binding capacity of 'kringle' 4. Esterification of carboxyl groups led to the loss of the lysine binding capacity which, however, was partially restored after demethylation at alkaline pH. A partial loss of the binding capacity was also observed after iodination, probably due to unspecific modification of histidine [16].

377 The lysine binding site is thought to consist of oppositely charged sidechains of two amino acid residues enabling electrostatic interactions with both terminal, functional groups of ¢o-aminocarboxylic acids. In order to guarantee the lysine binding properties it is essential that the 'kringle' structure be maintained by its three disulfide bridges. This may be taken as an indication that the amino acid residues of a binding site are probably located in different loops of a 'kringle'. The results of the CNBr-treatment indicate that the left outer loop of 'kringle' 1 is apparently n o t important for the lysine-binding property. However, its loss u p o n cleaving the right outer loop of 'kringle' 4 shows that part of the binding site is to be assigned to the sequence within this particular region of the 'kringle' structure. Specific chemical modifications allowed the exclusion of arginine as a possible participant in a binding site and also of lysine in 'kringle' 1. In the case of 'kringle' 4 the result of lysine modification can be interpreted in two ways: whilst it is feasible that the chemical modification of lysine introduces steric hindrance, it is also possible that in 'kringle' 4 a lysine residue does contribute to the binding site. Tyrosine was also excluded for reasons mentioned before. The amino acids remaining as potential participants in a lysine-binding site are, therefore, histidine and aspartate or glutamate. For the tentative localization of a binding site within the primary structure of a 'kringle' the following assumptions were made: Binding sites of the same class (for instance of low affinity) also have the same chemical nature, implying that in different 'kringles' always the same amino acids are involved. It was further assumed that each of the 'kringles' 1, 2, 3 and 4 possess a lysine-binding site [6]. As functionally and structurally important centers one might expect them to occur within evolutionary conservative sequence regions. On inspecting the known primary structure of t h e five plasminogen 'kringles' and considering experimental results and assumptions mentioned above one finds that only histidine occurring in all five 'kringles' in position 31 can meet these requirements. In addition, in 'kringles' 1, 2, 3 and 4, position 54 is occupied by an aspartate residue (Table I). In 'kringle' 5 which, according to experimental evidence [5], is apparently devoid of a lysine binding site, aspartate appears in the same position relative to the preceding half-cystine residue in the right inner loop. However, compared with the other 'kringles' the primary structure of 'kringle' 5 contains one more residue (Phe-36) in the right outer loop and compared to 'kringles' 2, 3 and 4 an additional residue in the right inner loop. It is possible that these differences in the primary 'kringle' structure create a steric constellation unfavourable for the binding of ¢o-aminocarboxylic acids. In the model which we would like to propose for a lysine-binding site, the protonated imidazole nitrogen of His-31 interacts with the dissociated carboxyl group of ~-aminocarboxylic acids (lysine, 6-aminohexanoic acid, etc.), whereas their p r o t o n a t e d e-amino function interacts with a dissociated carboxyl group, most likely that of Asp-54. The steric requirements of a binding site in the protein are given by the specific folding of the polypeptide chain in the 'kringie' structure, maintained by its disulfide bonds.

378 TABLE I S E Q U E N C E P O R T I O N S IN O N E L E T T E R C O D E O F T H E R I G H T O U T E R A N D I N N E R L O O P S OF T H E ~ K R I N G L E S ' 1 - - 5 [5] Right outer loop: 22 C Q K W S S

T

S

P

2

C

3 4 5

'kringle' 1

'kringle' 1

31

40

Q

A W D

S

Q

S

P

R P A H

C

Q

H

W S

A

Q

T

P

T

H

D

R

--

T

P

E

N

F

P

C

K

N

L

D

E

N

Y

C

Q

S

W S

S

M T

P

R

H

Q

K

--

T

P

E

N

Y

P

N

A

G

L

T

M N

Y

C

Q

D W A

A

Q

P

R

H

S

I

F

T

P

E

T

N

P

R

A

G

L

E

K

Y

E

i

right inner loop: 50 54 C

F -Y --

S I

P P

A S

T K

H F

P P

S N

E K

G N

L L

E K

E K

N N

6O

2

C

R

N

P

D

R

E

L

R

--

P

W

3

C

R

N

P

D

G

K

R

A --

P

W

4

C

R

N

P

D

A

D

K

--

G

P

W

C

R

N

PiDiG

N

V

G

G P

W

5

R G

This c o n c e p t is in principle applicable to the low affinity sites as well as to the single site of high affinity, if the possibility is considered that the intensity of electrostatic interactions of a binding site may be influenced by charged groups of other amino acids in its vicinity. We thank the Central Laboratory of the Blood Transfusion Service, Swiss Red Cross, Berne, for the generous supply of human plasma. This research was made possible by grant 3.118-77 of the Swiss National Science Foundation. References 1 2 3 4 5

M a r k u s , G., de Pa~quale, J.L. a n d Wissler, F.C. ( 1 9 7 8 ) J. Biol. C h e m . 2 5 3 , 7 2 7 - - 7 3 2 Markus, G., E v e m , J.L. a n d H o b i k a , G . H . ( 1 9 7 8 ) J. Biol. C h e m . 2 5 3 , 7 3 3 - - 7 3 9 Marlins, G., Priore, R.L. a n d Wisslar, F.C. ( 1 9 7 9 ) J. Bioh C h e m . 2 5 4 , 1 2 1 1 - - 1 2 1 6 Ricldi, E.E. a n d O t a v s k y , W.I. ( 1 9 7 5 ) Eur. J. B i o e h e m . 59, 4 4 1 - - 4 4 7 S o t t m p - J e n s e n , L., Claeys, H., Zajdel, M., P e t e r s e n , E. a n d M a g n u s s o n , S. ( 1 9 7 8 ) in Progress in C h e m i c a l F i b r i n o l y s i s a n d T h r o m b o l y s i s ( D a v i d s o n , J.F., R o w a n , R.M., S a m a m a , M.M. a n d D e s n o y e r s , P.C., eds.), Vol. 3, pp. 1 9 1 - - 2 0 9 , R a v e n Press, N e w Y o r k 6 L e r c h , P.G., Rickli, E.E., L e r g i e r , W. a n d Gillesscn, D. ( 1 9 8 0 ) Eur. J. B i o c h e m . 107, 7 - - 1 3 7 W i m a n , B. a n d CoHen, D. ( 1 9 7 8 ) N a t u r e 2 7 2 , 5 4 9 - - 5 5 0 8 Rickli, E.E. a n d C u e n d e t , P . A ; ( 1 9 7 1 ) B i o c h i m . B i o p h y s . A c t a 2 5 0 , 4 4 7 - - 4 5 1 9 Rickli, E.E., L e r g i e r , W. a n d Gillessen, D. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 3 9 , 4 7 - - 5 0 10 Bellin J.S. a n d Y a n k u s , C.A. ( 1 9 6 8 ) A r c h . B i o c h e m . B i o p h y s . 1 2 3 , 1 8 - - 2 8 11 Miles, E.W. a n d K u m a g L H. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 2 8 4 3 - - 2 8 5 1 12 P a t t h y , L. a n d S m i t h , E.L. ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 5 5 7 - - 5 6 4 13 Butler, P . J . G . , Harris, J.I., H a r t i e y , B.S. a n d L e b e r m a n , R. ( 1 9 6 9 ) B i o c h e m . J. 1 1 2 , 6 7 9 - - 6 8 9 1 4 B r o o m f l e l d , C.A., R i e h m , J.P. a n d S c h e r a g a , H . A . ( 1 9 6 5 ) B i o c h e m i s t r y 4, 7 5 1 - - 7 5 9 15 Azari, P.R. a n d F e e n e y , R . E . ( 1 9 6 1 ) A r c h . B i o c h e m . B i o p h y s . 9 2 , 4 4 - - 5 2 16 Means, G.E. a n d F e e n e y , R.E. ( 1 9 7 1 ) in: C h e m i c a l M o d i f i c a t i o n s o f P r o t e i n s , p. 1 7 6 , H o l d e n D a y , San F r a n c i s c o

N

Y Y