Analysis of ligand, binding to kringles 4 and 5 fragments from human plasminogen

THROMBOSISRESEARCH53; 243-252,1989 0049-3848/89 $3.00t .OO Printed in the USA. Copyright (c) 1989 Pergamon Press plc.

All rights reserved.

ANALYSIS OF LIGAND, BINDING TO KRINGLES 4 AND 5 FRAGMENTS FROM HUMAN V.V.NoVokhatnY,

PLASMINOGEN

Yu.V.Matsuka,

S.A.Kudinov

Institute Of Blochemistrr, Academy Of Sciences Of the Ukrainian SSRt Kiev, USSR (Received 7.8.1988; accepted in revised form 11.11.1988 by Editor B. Wiman)

ABSTRACT The interaction of the isolated krinBles 4 and 5 from human Plasminogen with 6-aminohexanolc acid, PentYlamine, pentanoic acid and areinine has been quantitatively characterized by scanning calorimetrv and fluorescent sPeCtroscOPY. It has been found that the lldands with the XlosltivelY charged BrouXj have a good bindlna ability while Pentanoic acid in ComPariSon with 6-aminohexanoic acid being devoid of amino drOUP does not interact with the krin&!leS under study. The POSitlVelY charged group of the 1iBand is Suggested to SliaY a crucial role in ligand binding with the lrsine-bindins site.

The nonvrotease raart of ulasmlnogen molecule contains five homologous triple-loop Structures, the kringles. It is known that kringles 1 and bind 4 are able to lysine and Other O-aminucarboxYlic acids flZ!l.Most of these amino acids exhibit the uotent antifibrinolrtic activity. LYSlne-binding sites Play a ker roie in regulation of PhrsioloBical fibrinolvsls since they are resuonslble for the interaction of Plasmin with fibrin and dz-antirllasmln I31, Information on the binding site of krinsle 5 is Quite CfrntradiCtor~. This domain is able tO interact With benzamldlne';erharose C41, aminohexrl-eeuharose I53 and arginrl-SePharose I63 that vermitted ascriblnll the rlresence of the benzamrdine-blnding site and AH-site to krinale 5. But at site1 arginine-binding the same time krinale 5 1s not bound to lvsine-gerharose Cll, it to be deVOld of the that was the to Consider reason Ker

words: Plasminoden,

lYsine-binding 243

site, krinble

4,kringle 5.

244

LIGAND-KRINGLE INTERACTION

Vol. 53, No. 3

that isolated ShOWn IYSine-bindind Site. HOWeVer, we have krinale 5 interacts with 6-aminohexanoic acid in a solution [71. Furthermore, the fibrin-miniPlasminoaen interaction is inhibited br 6-aminohexanoic acid C&93. These facts lead to a SUddeStiOn that krinale 5 contains the lrsine-blndind site with a broad structural to definite SPecificitY which liaand is related Peculiarities of this domain. It is considered that on the binding of ligands with a lysine-binding site there occur eleCtrOStatiC interactions, in which Asr, 56 and Ar8 70 form electrostatic &airs with amino- and carboxrlic BrouPs of the lidand COmPlementarY to them [loI. It means that both cationic and anionic centers are necessary simultaneOuSlY for the liaand binding.' The develonment of IlOtionS on the lrsine-binding Site Structure Permits Understanding more Profoundly the possible mechanism of the lidand interactions with krinales and ConsequentlY the mechanism of Protein-Drotein comnonents of the fibrinolvtic interactions of a 11d haemocoaBulation systems. At present a probable structure of the lrsine-binding Site represents a Cavity which is formed bY the hrdroohobic amino acid residues with well defined negative charge inside it li7,11r123. Proceedind from such a structure it becomes evident that in interaction of lllland with the Irsine-binding site the Positivelr charged group of the liaand should Play a crucial role. calorimetrv The methods of scanning and fluorescent sPectroscoPr have been used In this paper for verification of the last SUYDoSitiOn and alS0 for comnarative characteristics of Iisland svecificity of the lusine-blndina sites of krinales 4 and 5.

Human rlasminogen was rrurified from Cohn Fraction III br affinity chromato%ranhY according to Deutsch and MertZ 1131. Krinllle 4 was isolated from human PlaSmlnOBen br elastase didestlon of the ZYmOlen as described elsewhere C141. The heavy chain of Plasmin was obtained from rllasmin br Selective reduction and 8cetYlation of its interchaln disUlPhldes and isolated bv affinity ChrOmatOaraDhY on lrsine-SeDharose accordina to El51. Krindle 5 was orePared bY elastolrtic digestion of heaVY chain of plasmin and DUrifled bY affinity chromatosranhr on luslneSenharose as described earlier Cl4l. The homoaenertr of the StUdled fragments was checked br sodium dodecrl sulnhate PolracrYlamide Be1 electrorhoresis as described in Cl6). The followind materials were obtained from the indicated commercial sources: elastase (Sigma Chemical CO.~St.LOUislYO~USA), nentylamine, Pentanoic acid, aralnine and Gaminohexanoic acid (Fluka, Fuchs, Switzerland). Calorimetric measurements were carried out in a differential adiabatic mlCrOCalOrlmeter DASM-1Y 1173 at a scan rate of 2 KImin with 1 ml cells. Degassing during heating was Prevented With the use of extra Constant pressure of 1.5 atm. over the liquids in the cells. Protein concentrations were 0.2-0.4 ma/ml. Protein SOlUtiOnS for CalOrimetrY were equilibrated bY dialYSiS against a 150 mM sodium PhOSPhate buffer, PH 8.0. When the effect of

Vol. 53, No. 3

LIGAND-KRINGLEINTERACTION

245

Dentvlamine, Pentanoic acid and 6-aminohexanoic acid on test kringles was analyzed, Droteins were dialyzed against 100 mM sodium PhOSDhate buffer, DH 8.0 containing 50 mM of tested ligand. Fluorescence titrations of kringles 4 and 5 were Performed with a "Hitachi" MPF-4 fluorescence SDectroDhotometer in 50 mM For all SamDles, Tris-HCl, DH 7.4, 50 mM NaCl buffer at 25% an excitation Wavelength of 295 nm and a Slitwidth of 6 nm were used. The emission SPectra were monitored at 330 nm with a 6 nm Slitwidth. Protein concentration was ~J.IM, Protein solutions were Placed in 4-ml quartz CUVetteS and titrated with 3-6~41 aliquots of concentrated lieand solution. Final concentrations of the ligands used in this exYeriments were in the range of for PentYlamine 0.01-l mM for 6-aminOheXanOiC acid* 0.1-10 mM and 0.01-10 mM for arginine. Corrections for the inner filter effect were unnecessary at the excitation wavelength and at the s01ut10ns used in this study. concentrations of ligand data were analyzed according to Fluorescence titration Scatchard 1181. ECUITS been Shown earlier that the interaction of It has 6-aminohexanoic acid with the seDarate domains of the PlasminoBen sites results in an molecule containing the lrsine-binding increase of their temDerature stability C14,191.This effect is SDecific and DrOPortiOnal to the binding strength. This aDDroach was used to investigate the interaction of various ligands with isolated krrneles 4 and 5. Fig.1 presents the melting curves of the test kringles reflectin6' the interaction of lisands with the lrsine-binding sites. Table 1 lists the transition temperatures. In the case of krin8le 4 which COntaiIlS a real lvsine-binding site the maximal temPerEttUre shift is Observed at its melting with 6-aminohexanoic acid. PentYlamine, however, causes the stabilization of the kringle 4 Structure bY 6 oc. This fact points out the interaction of this COmPOUnd with the lrsrne-binding site. But at the same time stability of kringle 4 was unaffected bY Dentanoic acid.Thus, the exDerimentS show that Dositivelr charged drouD is important in binding of lisand with the lrsine-bindins site of kringle 4. Besides, negatively charged 8rouD is .neCeSSarY for

such

an

interaction

which

aPYears

to

enhance

the

bindlne

Strength. Quite different Picture is Observed in the case of the kringle 5. ADProximatelY the same temPeratUre shift Droceeds both at the melting of kringle 5 in the Presence of 6-aminohexanoic acid and at its melting in the Presence of Dentrlamine ill,5and 10.3 act resDectivelY1. Stability of kringle 5 as well as of These results krindle 4 was unaffected bY Pentanoic acid. drouD is required for indicate that only DOSitiVelY charged interaction of ligand with the IYSine-binding site of the fifth kringle. ViSUallY that Thus, the calorimetric results demonstrate Dositivelr charged drOUP Of the lidand PlaYS a Crucial role in the interaction with the lusine-binding Site.

LIGAND-KRINGLE INTERACTION

246

Vol. 53, No. 3

KAINGLE 4 A

FIG.1 TemDerature krineles 4 and 5. presence of 50 mM Dentrlamine; (41 in

80

40

0

4

deDendence of Partial heat CaDaCities of (11 in the absence of ligands; (2) in the Dentanoic acid; (31 in the Dresence of 50 mY the Presence of 50 mM 6-aminohexanoic acid. TABLE 1

Transition Temperatures of Krinsle with Tested Ligands . Melting

4 and

KrlnPle

5 at

K4

K5

150 mM sodium DhosDhate ,DH 3,O

57.0

53.0

100 mM sodium DhOSDhate,PH 8.0 +50 mM Dentanoic acid

57.0

53.4

100 mM sodium DhOSDhate ,DH 8.0 +50 mM Dentrlamine

62.4

63.3

100 mM sodium Dhosuhate t DH 8.0 +50 mM 6-aminohexanoic acid

71.0

64.5

EXPERIMENTAL CONDITIONS

Transition temDerature the diVen temDeratures

is

given in OC. The is 20.2 oc.

absolute

error

Their

of

Vol. 53, No. 3

LIGAND-KRINGLE INTERACTION

247

Fluorescence snectrosconY was used to assess the 4uant1tative characteristics of the liaand b1nd1n6, The fluorescence 1ntens1tr narameters under study were solely due to trYPtoPhan fluorescence, since excitation at 295 nm and em1Ss1On at 330 nm were emPlOYed in the exrlerlments. A fluorescence 1ntensitY increase resulted from the interaction. Of 6-aminoheXano1c acid, Pentvlamine and arBin1ne with KrinBle 4, whereas a decrease 1n this Lsarameter was found on interaction of kr1nele 5 with the same 11bands. No fluorescence intensity alterations were observed UPon lnteractlon of Pentanolc aC1d with krinale 4 or kr1nBle 5. Such different intrinsic fluorescence chandes may be connected with the aiteratlons 1n the structure of these kr1ndles. In PartiCUlart in the fourth kr1ndle trYPtoDhan residues 1s situated at PoS1tiOn 71, while 1n the fifth kringle tYrOS1n 1s situated at the same POS1t1On [il. The titration nroflles of krlnales 4 and 5 1n the nresence of the varY1nB concentration of lidands indicate site-sDec1fic liaand bindind. The Scatchard Plots of these data (Fig.2 and F1S.3) dive calculated dissociation constants being summarized 111. Table 2. As seen from Table 2, dissociation constant of krindle 4 for 6-am1nohexanoic acid is [email protected] value 1s 1n 8ood agreement with E2,201. The d1SSoC1at1On constants established earlier h1tilherr d1SsoC1at1On constant for nentYlam1ne 1s two orders 3.3 mM. The d1ssoc1atlon constant for arB1n1ne 1s still higher, 12.1 mM. But 1t 1s of 1ntereSt t0 note that both PeIltYlam1ne and ard1n1ne Interact sX~ec1f1callr with kr1nole 4. However, these interactions are to a considerable extent weaker 1n comYar1SOn with 6-aminohexanoic aC1d. In the case of kr1Iible 5 all three 11gands with a POS1t1VelY charged Brouv YOSS~SS a dood b1ndinB abilitY.ArB1n1ne 1s shuwn to have a maximal b1ndiI-M strength. The dissociation constant for it 1s 19JM. The dissociation constant for 6amlnohexanolc acid 1s

0

0.4

0.8 AF

1.2

Scatchard FIG.2 Plot of the fluorescence titration of kr1nBle 4 with 6 - am1nohexano1c acid (OL ar81n1ne (e 1 and iA l. rentYlam1ne

LIGAND-KRINGLE INTERACTION

248

0

0.1

0.2

0.3

OA

AF

Vol. 53, No. 3

FIG.3 Scatchard plot of the fluorescence titration of krinBle 5 with 6 - aminohexanoic acid (0 1, arginine (0) and YentYlamine (A 1.

TABLE 2 Dissociation Constants of LIGAND

KrinBles 4 and 5 K4

for

Tested

Ligands.

K5

6-aminohexanoic acid

27 x lo-6bi

64 x lo-%

PentYlamine

33 x lo-4Y

163 x lG-%d

121 x 10_4M

19 x lo-6M

no binding

no binding

Arainine Pentanoic acid The

relative error of the given Kd values did not exceed 12 % .

somewhat higher, 64& And PentYlamine has a minimal binding ability. The dissociation constant for this lidand is 163~~Y. Thus, these results indicate directly that the kringle 5 binding site has broad ligand SPecificitY.

It is SUpDOSed from the outset of the investiaations of the lrsine-binding site structure that the both Yositive and negative charges of the liPand are essential for the ligand binding. In the works of Lerch and Rickli I211 and Trexler et aLI all investigations were aimed at the determination of amino acid

Vol. 53, No. 3

LIGAND-KRINGLE INTERACTION

249

residues Which are comniementarr to the PoSitiVelY end negetivelr charded EIrOUPS Of the llgand. It has been shown br chemical modification that Ard 70 and ASP 56 are essential for the bindlncl of lidands bY kringle 4 ClOl.These results were eXtrarOlated on other krlngles and on the basis of the knuwn Sequence of the hOm01080US krlndles of PlesmlnoBen it was determined whether a kringle may or may have not a homologous lrsine-binding site. In Darticular, according to this reesonin8 kringle 3 and krindle 5 would not be expected to have a lusine- binding site since As.1156 was replaced br a 1Ysine residue in kringle 3 and both essential bY neutral amino acids 11-I were rePlaced Charged residues krinE!le 5. Doubt were thrown uuon the Trexler et al. suvL~ositions when it became evident that krindle 5 alS0 has a bindind Site [4-71. that ASP 54 is eSSentia1 for In edditlon, it has been Shown fibrin and 6-eminohexanoic acid affinitu of krlnble 1 [223.Thus, the annroach aCCOrdinL;Ito which the Presence or the absence Of the binding site is determined OnlY bu amino acid residues in Bositions 56 and 70 of the krinole sequence is not quite correct, number of Works on After the Publication of a NMH-SPeCtrosCOPY devoted to investi&!ations of the lrsine-bindind site structure it became evident that esuivalence of charded droUPS of the lidand should be revised. Accurdina to these Works C11,12,23-251as well as to our fluorescence investitietions C71 the Structure of the luslne-brndlng site of krlnsles i, 4 and 5 is a CaVitY which is formed bY hrdrorhobic amino acid residues. A well defined negative charge is localized at the bottom of this Cavity. Proceeding from this structure natural SuvPosition arises that first of all lidand should enter the cavity with its POSitiVelY charged end and only then, probablv, the negatively charged droUY of ligand should be closed with uositivelr charged amino acid residue forming the cationic center of the lysinebinding site. In this paper We rerresent the results of iIlvestiBations aimed et verification of this SunL~osition. The fourth and fifth kringles of the nlasminosen molecule were USed for the lnvesti8ations. KrinrJle 4 was shown in a numerous works to carry a real lrsine-binding site. At the same time infOrmatlOn about bindins! Site Of kringle 5 is rather scanty and any data concerninf?! its binding 9roPertles are of great interest. The model comvounds tested were yentanoic acid and rentylamine which in ComParison with 6-aminohexanoic acid are devoid of amino 6rouP and carboxYlic JrouP, resnectivelr. In addition, these compounds are soluble in aclueous solution in contrast to hexanoic acid and hexvlamine which, strictly Sneaking, should be Used. Ardinine was taken as a lidand containing Buanidrl group. bu scannin&‘ calorimetrr Pentanoic acid as found and fluorescence SPeCtrOSCOPY was not bound to lrsine-binding sites Of krinclles 4 and 5. At the same time rentYlamine interacts both with kringle 4 and krinllle 5. This leads to the COIiClUSiOn that rosltivelY charged droUD of the Mand Plars a crucial role in Mend binding. But it is imPortant to note that the bitlding strength of nentrlamlne with krinale 4 and krin83le 5 1s different. The dissociation constant for binding of 0entYlamine with orders higher in comnarison with kringle 4 is two

250

LIGAND-KRINGLE INTERACTION

Vol. 53, No. 3

6-aminohexanoic acidt that indicates that the CarbOXYliC group of lii3and Provides stabilization of the binding of 6-aminohexanoic acid to kringle 4. On the ligand binding of kringle 5 the carboxylic group of the ligand Plays a negligible role since no essential distinctions in dissociation constants for the binding of 6-aminohexanoic acid and uentrlamine can be noted. In our view, this conclusion is very important since it makes Possible to explain a broad ligand SPeCifiCitY of the binding site of kringle 5. Indeed, it becomes evident WhY kringle 5 is able to interact with different affinity sorbents. All affinity sorbents mentioned above have a Dositivelv charged BrOUr at the end of the bulky hrdrorhobic base. Aminohexrl-Serharose has amino Brour, while benzamidine- and arginine-SePharose have guanidY1 BrouPs. In our eXPerimentS the dissociation constant for the binding of areinine with kringle 5 is some lower than for 6-aminohexanoic acid (19 and 64$4, resPeCtivelYl.The maximal binding ability of arginine in case of krinlle 5 appears to be connected with more strong charge of its Buanidul Brour in ComDarisOn with 6-aminohexanoic acid amino ProuP. Thus, our results demonstrate the Paramount imnortance of UositivelY charged group of the ligand at its binding with the lYSine-binding site. It should alS0 be indicated that our data are in agreement with results obtained br Christensen according to which the binding Site of KrinBle 5 Prefers li8andS not carrying a free carboxrlate function, In our orinion~ the information obtained is quite important for understanding the mechanism of the Plasmin(oBen1 interactions with fibrin. Firstly, it becomes evident that kringle 5 may Play an important role in such interactions. Secondlr, the Structure Of Plasminogen-binding site on fibrin molecule may be DroDOSed. Such a site should not have obli8atOrY the DoSitiVelY and negatively Charged amino acid residues. Only lrsine or art?inine residue is suite enough for it. But it should be emphasized that this YOSitiVelY charged residue must be arranged in such a site of POlYPeYtlde backbone which is situated near the Protein surface.

We thank S.E.Milenko for competent tYPing V.A.ShUleZhkO for technical assistance.

the

manuscrint

and

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