Isolation, purification and 1H-NMR characterization of a kringle 5 domain fragment from human plasminogen

Biochimica etBiophysica Acta 912 (1987) 254 269 Elsevier

254

BBA32707

Isolation, purification and I H - N M R c h a r a c t e r i z a t i o n of a kringle 5 d o m a i n fragment from human plasminogen T h e r e s a T h e w e s , V a s u d e v a n R a m e s h , E l c n a L. S i m p l a c e a n u a n d M i g u e l Llinfis Department of Chemistry, Carnegie-Mellon University, Pittsburgh, PA (U.S.A.) (Received October 20 1986)

Key words: Plasminogen; Kringle 5; Benzamidine; Protein NMR; Miniplasminogen; NMR, 1H

A scheme is proposed for generating the intact Val-448-Phe-545 polypeptide of human plasminogen which contains the fifth kringle domain of the plasmin heavy chain. The procedure is based on a pepsin fragmentation of miniplasminogen and involves the purification of the kringle 5-containing fragment by gel filtration and ion-exchange chromatography. The final product is characterized by amino acid analysis, Nand C-terminal analyses, and high-resolution tH-NMR spectroscopy at both 300 Mltz and 611 MHz. We detect a (40:60%) Asp/Asn heterogeneity at site 452 of the Glu-plasminogen molecule. In the conventional kringle numbering system, the kringle 5 domain extends from Cys-1 to Cys-80, which corresponds to Cys-461 to Cys-540 in plasminogen. A preliminary tH-NMR characterization of kringle 5 focuses on the global conformational features of the polypeptide. Assignments are given for a number of resonances, including the Tyr-72, the His imidazoles' and the Trp indoles' spin systems. Comparison with human plasminogen kringles 1 and 4 shows that the kringle 5 conformation is highly structured and very similar to that of the homologous domains. This conservancy is particularly striking in the environment surrounding Leu-46 and in the overall features of the aromatic spectrum. There are some differences, particularly in the buried His-33 imidazole group, whose H2 resonance is shifted to 9.67 ppm. A preliminary study of benzamidine-binding shows that the ligand interacts weakly (K a = 1.7 mM - t ) mainly through the amidino functional group. Trp-62 and Tyr-72 are significantly perturbed by benzamidine, suggesting that these residues are part of the ligand-binding site.

Abbreviations: BASA, p-benzylaminesulfonic acid; COSY, two-dimensional chemical shift correlated spectroscopy; DEAE-Sepharose, diethylaminoethyl-Sepharose; HPLC, highperformance liquid chromatography; K a, ligand-kringle association constant; K1, kringle 1 (Cys-81-Cys-161); K4, kringle 4 (Cys-357-Cys-436); K5, kringle 5 (Cys-461-Cys-540); K1 +2 + 3, Kringle 1 + 2 + 3 (Tyr-79-Val-337); pH*, glass electrode pH reading uncorrected for deuterium isotope effects; pK*, pK a determined from acid/base titrations in 2H20 uncorrected for deuterium isotope effects; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. Correspondence: M. Llinhs, Department of Chemistry, Carnegie-Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, U.S.A.

Introduction Plasminogen is the 790 amino acid zymogen of plasmin, a blood plasma serine proteinase. This precursor is activated to the two-chain enzyme by cleavage of the Arg-560-Val-561 peptide bond and release of a 76-residue amino-terminal peptide. Plasmin's proteinase active site is localized within the C-terminal light chain [1]. The N-terminal heavy chain contains five homologous regions, kringles 1 to 5, with a characteristic triple disulfide bond pattern. This 'kringle' [2] loop structure has been shown to define both a structural and a

0167-4838/87/$03.50 © 1987 Elsevier Science Pubfishers B.V. (Biomedical Division)

255 folding domain [3-5]. It is found in several proteins, including prothrombin [6], urokinase [7], haptoglobin [8], tissue plasminogen activator [9] and factor XII [10]. Studies have shown that kringles from several sources bind lysine and other t0-amino acids [11-16]. It has been proposed that the kringles are responsible for anchoring plasminogen to fibrin blood clots through their binding sites [1,17]. Once activated to plasmin, the serine proteinase portion of the molecule is involved in fibrinolysis. Elastase digestion of plasminogen (Fig. 1, black arrows) yields four fragments: preactivation peptide (residues 1-76), K1 + 2 + 3 (kringle 1 + 2 + 3, residues 77-353), K4 (residues 354-439), and miniplasminogen (residues 442-790 = K5 + light chain) [1]. The K1 + 2 + 3 fragment can be further digested with chymotrypsin [11,18] or Staphylococcus aureus V8 proteinase [19] (Fig. 1, circle) to yield K1. In previous papers, we [19-22] and Williams' group at Oxford [23,24] have reported on the solution structures of K1 and K4 based on highresolution 1H-NMR spectroscopy studies. The assignment of aromatic resonances in the human plasminogen K4 spectra has been deduced on the basis of side chain chemical modification studies [21,23,25] and by comparison of various homologues [21,22]. The assignment of the K1 reso-

K4 Fig. 1. Peptide backbone outline of the human plasminogen molecule showing all disulfide bonds [1]. Cleavagesites for the various proteinasesare indicated: pancreatic elastase (,,,4), S. aureus V8 proteinase (O) and pepsin (E::~). Asterisks (*) denote the streptokinase activation site and the interchain disulfide bond reduction sites.

nances is based, for the most part, on comparative spectroscopy with K4 [19,21]. In 28 of 80 positions all five of the human plasminogen kringles are identical; 13 of these 28 residues are Pro, Gly or Cys [1], which can strongly influence the secondary and tertiary structures. The overall similarity in amino acid sequence suggests a uniform folding pattern for the kringles. The N M R studies of K1 and K4 substantiate this proposal [21]. Although both K1 and K4 contain a lysine-binding site [11-14], this does not seem to be the case for fragment(s) containing K5. However, this kringle does bind benzamidine [26] and probably arginine [27]. The variation in binding specificity suggests that K5 may play a unique role in fibrinolysis. This prompted us to initiate structural N M R studies on this fragment vis h vis the conformational and functional characterization of the K1 and K4 homologues, which are currently under way [19,22,24]. Two methods for K5 isolation have been reported. The first [26,28] involves activation of the miniplasminogen to miniplasmin by streptokinase-initiated cleavage of the Arg-560-Val-561 peptide bond (Fig. 1, asterisks). This is followed by reduction and alkylation of the two interchain disulfide bonds between Cys residues 547 and 665 and between Cys residues 557 and 565. The K5 product thus isolated is composed of residues 442 to 560. The second reported method of K5 isolation involves a pepsin cleavage of miniplasminogen [5], presumably after Phe-545 (Fig. 1, open arrow). The K5 fragment which is isolated with this procedure would thus be composed of plasminogen residues 442 to 545. This paper presents an improved pepsin digestion and purification scheme for plasminogen K5, a characterization of the isolated kringle fragment, and preliminary results of our ~H-NMR studies at 300 MHz and 611 MHz. These initial N M R studies verify that K5 binds benzamidine with K~ --- 1.7 mM-1. Materials and Methods B i o c h e m i c a l procedures

Plasminogen was isolated from aged citrated human blood plasma (a gift from the Central Blood Bank of Pittsburgh) by affinity chromatog-

256 raphy on lysine-Sepharose [29,30]. The gel matrix was prepared according to the methods of Cuatrecasas et al. [31]. Elastase was extracted and purified from an acetone powder of porcine pancreas (Trypsin 1-300, Nutritional Biochemicals Corp.) following the method of Shotton [32]. Miniplasminogen was isolated from an elastase digest of plasminogen as reported by Sottrup-Jensen et al. [1]. Kringle 5 was obtained from a pepsin digest of miniplasminogen according to the procedure described below. Analytical-grade buffer salts were used throughout. Chromatographic gels were purchased from Sigma. A 0.5 m g / m l pepsin (U.S. Biochemicals) solution was prepared in 0.05 M glycine buffer (pH 2.5). Miniplasminogen was dissolved in 0.05 M sodium phosphate buffer (pH 7.4) to 10 m g / m l ; the p H was then lowered to 2.5. Pepsin was added to the miniplasminogen solution to give a 1 : 400 (w/w) ratio of pepsin/miniplasminogen. After 75 min incubation at 25 ° C the reaction was quenched by raising the pH to approx. 8.0 with 1.0 M NaOH. The remaining steps of the isolation procedure were completed at 4 ° C. The reaction mixture was dialyzed (membrane cut-off M r --- 3500) overnight against distilled water and lyophilized. It was subsequently applied to a Sephadex G-75 column (120 x 2.6 cm) and eluted with 0.1 M ammonium bicarbonate with 0.02% sodium azide (pH 8.0) (Fig. 2A). The K5-containing pool was localized by 1H-NMR (peak III), lyophilized, and applied to a DEAE-Sepharose column (50 × 1.6 cm) which had been equilibrated with 0.05 M Tris-HC1 buffer (pH 8.0). After washing extensively with the buffer, a 500 ml, 0-to-1 M NaC1 gradient (in the Tris buffer) was applied. The first peak from this column (Fig. 2B) contained unidentified fragments of approximately the same M r as K5. The second peak contained K5 exclusively, a fact which was established by 1H-NMR criteria and amino acid composition, and was subsequently applied to a Sephadex G-50 column (120 × 1.6 cm) for desalting. The final product was stored as a lyophilized powder. About 0.50-1.00 g of miniplasminogen was digested per batch. The amount of K5 obtained by this purification is approx. 10% of the theoretical yield. The progress of the digestion was monitored by polyacrylamide gel electrophoresis (PAGE) and

by SDS-PAGE, under both reducing and non-reducing conditions, using the LKB Multiphor apparatus and reagents as described in the LKB Application Note 306. Gels were poured using the GelBond PAG film/AcrylAide cross-linker system of FMC BioProducts (Rockland, ME); the destaining solution was acetic acid/methanol (10 : 45, v / v ) in water. Most of the analysis of K5 has been performed on the species isolated from the DEAE-Sepharose column with no additional fractionation. However, conventional electrophoresis shows that there are two differently charged species in this sample (Fig. 3, lane c) and a further purification step was implemented to characterize each of these constituents. The two-component K5 was reapplied to the DEAE-Sepharose column and eluted with a more shallow NaC1 gradient (0.15 to 0.30 M NaC1 in Tris buffer, p H 8.0; total gradient volume = 200 ml). The protein eluted as a single broad peak. The first few fractions and the last few fractions of the peak were pooled and desalted separately for further analysis. Protein samples were hydrolyzed for 24 h in 6 M HC1 at l l 0 ° C , in vacuo; the amino acid composition of the hydrolysates were determined using a Durrum D500 amino acid analyzer. N-terminal analyses were performed on the Applied Biosysterns 470A Protein Sequencer. The C-terminal amino acid of K5 was cleaved by carboxypeptidase A (Sigma) using the method of Ambler [33]. After digestion and precipitation of the protein, free amino acids of the supernatant were determined using the Durrnm D500 analyzer. N M R methods

Proton N M R spectra were recorded in the Fourier mode at 300 MHz with a Bruker WM-300 spectrometer and at 600 or 611 MHz using the spectrometer of the N I H National Facility for Biomedical Research at Carnegie-Mellon University. Conventional one-dimensional spectra were acquired with quadrature detection over a 16K data block. Spectral widths for the 300 MHz and 611 MHz spectra were 5000 Hz and 10000 Hz, respectively. Kringle 5 spectra were routinely accumulated in 2H20 after exchange of the labile hydrogen atoms for deuterium. The residual 1HZHO signal was irradiated for 2.3 s with low

257

decoupling power before acquisition. Spectra recorded on 1H20 solutions were obtained using the 1-1 hard pulse sequence [34]. Resolution enhancement was achieved by Gaussian convolution [35,36]. Chemical shifts are referred to the 3-trimethylsilyl[2,2,3,3-2H4]propionate signal assumed to resonate at - 3 . 7 6 6 ppm from the dioxane internal standard. For the acid/base titration studies, the p H * was adjusted with small additions of concentrated N a O 2 H or 2HCI solutions. Acid/base titration curves were derived from digital readouts of the corresponding spectral peak positions. When two curves crossed-over, the identities of the resonances were determined from their line shapes, assumed to be preserved across the resonance overlap point. Histidyl irnidazole p K * values were derived by fitting N M R data to the equation [37]:

~.+-

8.o

K~' + [ H + ] "

where K . is the dissociaton contant of the side chain, 8H÷, 8Ho and 6obs are the imidazolium, imidazole and observed chemical shifts respectively, and n is the Hill coefficient. Two-dimensional COSY [38] spectra were acquired at 300 MHz with 512 equidistant values of t~ and 320 transients added for each t 1 value. The spectral width was 3333 Hz, with a data block of 2048 addresses in the 82 dimension. Quadrature detection was implemented with appropriate phase cycling to select N-type peaks (Bruker Aspect 2000 Data Package). The residual 1H2HO signal was suppressed by low-power gated irradiation during the relaxation delay of 2.3 s which was introduced between scans. Before Fourier transformation, the t I dimension was zero-filled to 1K and multiplied by a sine bell in both dimensions. This results in a digital resolution of 3.255 Hz. The contour plot is shown in the absolute value mode after symmetrization. Benzamidine-binding was studied by adding small volumes of a concentrated, pre-exchanged solution of the ligand to the K5 sample. Several thousand scans were averaged after each ligand addition. Dilution effects were taken into account when calculating K a, the association constant for ligand-binding to K5 [14,39]. In the analysis, we

assume single-site binding as described elsewhere for BASA-binding to K1 [14]. Results

Purification of kringle 5 The pepsin digestion gives rise to the chromatograph shown in Fig. 2A. Fractions were combined into six pools, indicated by Roman numerals I-VI. Initially, SDS-PAGE experiments were performed under both reducing and non-reducing conditions. Under non-reducing conditions, the major component of pool I exhibits M r = 38000 ( M r of miniplasminogen) and the major component of pool II indicates M r ~ 27000 ( M r of the light chain of miniplasminogen). Under reducing conditions, both pools I and II give a major band at approx. 11 kDa, suggesting extensive cleavage in the light chain portion of the miniplasminogen. Under both reducing and non-reducing conditions, pools I I I - V all contained fragments similar in size to a kringle ( = 11 kDa). ~H-NMR spectra were recorded on pools I I - V after the proteins had been exchanged in 2H20 as

G-75 Sephodex

A

DEAE Sepharose

B

5.0

2.0

g ~

0 1.0

0.5

20

40

60 80 100 Fr(]ction Number

120

Fig. 2. Chromatographic profiles of a miniplasminogen pepsin digest. The K5-containing peaks are shown hatched. (A) Sephadex G-75 fractionation of the pepsin digest. (B) DEAE-Sepharose fractionation of peak III material of (A).

258

previously described. Only the spectra of pool III exhibited characteristic fingerprint signals observed in other kringles which have been studied by N M R [14,20,21,23]. It was obvious from the conventional PAGE (Fig. 3, lane a) that pool III contained more than one component. Appfication of pool IlI to a DEAE-Sepharose column with a NaC1 gradient elution yields the profile shown in Fig. 2B. There are two well-resolved peaks. The first corresponds to at least two contaminating species as determined by gel electrophoresis at pH = 9 (Fig. 3, lane b). :H-NMR identifies the second peak as a kringle fragment; it appears as two bands on conventional PAGE at pH ~ 9 (Fig. 3, lane c). When the two-component K5 was reapplied to the DEAE-Sepharose column and eluted with the shallow NaC1 gradient, a single, broad protein peak was eluted. The beginning and end of this peak were pooled separately; PAGE of these pools are shown in Fig. 3, lanes d and e, respectively. An amino terminal analysis was performed for each of these pools by running six or more cycles on the sequencer. The results of this sequencing verify that both of the pools are K5 and that there are two major variants. The species from the begin-

+

K°: Slotsa

b

c

d

e

Fig. 3. Gel electrophoresis of selected chromatographic fractions. Peaks refer to Fig. 2. Lane a, Sephadex G-75 column peak ili; lane b, DEAE-Sepharose column peak I; lane c, DEAE-Sepharose column peak II (K5); lane d, (Asn-(- 8) K5; lane e, (Asp-(-8) K5. Conventional 5.0% PAGE; Tris/glycine buffer, pH 8.9.

ning of the shallow gradient peak contains proteins with amino termini which correspond to Val-(-12) (~20%), L e u - ( - l l ) (~40%), and Leu-(-10) ( ~ 40%) with an Asn residue at position - 8 . The species from the end of the shallow gradient peak is characterized by amino termini which correspond to L e u - ( - 10) and Pro-(- 9) in about equal proportions, with an Asp residue at position - 8 . These results indicate multiple cleavage sites within the N-terminal chain of the kringle, which is not surprising considering the variable specificities of the proteinases: elastase cleaves preferentially after both Ala and Val residues and pepsin cleaves mostly after both Phe and Leu residues. The A s p / A s n heterogeneity at position - 8 of the N-terminal segment may explain the two bands seen on PAGE (Fig. 3, lane c). In approx. 60% of the K5-containing fragments this residue is an Asn, otherwise ( ~ 40%) this residue is Asp. At high pH one would expect the Asp-(-8) K5 species to have an additional negative charge: thus its retarded elution from a DEAE-Sepharose column and faster migration in conventional PAGE experiments. Since reaction at the C v amide group is unlikely, the heterogeneity at position - 8 probably arises from genetic variability in the blood plasma pool. Site - 8 corresponds to site 452 of the intact Glu-plasminogen molecule [1]. Carboxypeptidase A digestion of the protein liberates only phenylalanine. This is consistent with the expectation of no significant cleavage of the Pro-83-Ser-84 bond by this enzyme [33]. These results confirm specificity for the pepsin proteolysis at the plasminogen Phe-545-Asp-546 link, i.e., leaving Phe-85 as the kringle fragment C-terminus. Also, since no other free amino acids are released following the carboxypeptidase A digestion, no additional proteinase cleavage sites are expected to be present within the kringle structure. The amino acid analysis of the purified kringle fragment (Table I) compares well with that expected from the kringle 5 primary structure (Fig. 4). Deviations, for the most part, can be attributed to heterogeneity in the N-terminus. Clearly, we have isolated a K5 fragment composed of human plasminogen residues 448-545 with some variability at the N-terminal (non-kringle) tail.

259

TABLE I

50~ xrlj .

A M I N O ACID ANALYSIS OF K R I N G L E 5

.

.

.

,\p,

Amino acid "Expected b Observed c N-terminus d C-terminus ~ analysis analysis (%) (%) Asx Thr Set Glx Pro Gly Ala Cysr Val * Met Ile Leu * Tyr Phe His Lys Arg Trp

12 9 3 9 11 9 6 6 5 1 1 4 5 3 2 4 6 2

12.0 8.9 2.7 9.4 13.1 9.1 6.2 1.7 4.4 1.0 1.0 3.0 5.3 3.0 2.1 4.1 6.5 -

20

12

10 68

c~)

-S'

100

a Residues that yield lower-than-expected values because of multiple elastase cleavage sites at the N-terminal peptide chain are denoted by an asterisk (*). b Expected amino acid composition based on the assumption of a homogeneous N-terminus sequence, starting with Val( - 12) as shown in Fig. 4. c Observed values are normalized to Met = 1. d See Methods. As determined by free amino acid analysis following carboxypeptidase A digestion. t The Cys content analysis is not likely to be accurate, as the K5 sample was neither reduced nor carboxymethylated prior to hydrolysis.

~H-NMR characterization of kringle 5 The complete 611 MHz proton spectrum of K5 dissolved in 2H20 is shown in Fig. 5. Specific features of the spectrum suggest that the resonance positions are influenced by secondary and tertiary structural constraints. These features include: (a) the spread of aromatic signals ranging from approx. 5.0 ppm to approx. 9.6 ppm, (b) the presence of resonances from backbone ~t-protons shifted to lower fields than the aH2HO signal, (c) methyl groups resonating up to approx. - 1 ppm. Doublets at 0.38 and - 1 . 0 4 ppm (Fig. 6C) belong to the same Leu side chain spin system, as they are coupled to a proton giving a signal at 1.06 ppm (couplings of the CH ~'~' protons to the CH ~

Fig. 4. Kringle 5 of human plasminogen: outline of the primary structure. The residues are numbered starting from the first half-cystine (Cys-1) so that amino acids within the aminoterminal peptide tail are labelled zero or with negative numbers. The amino acid sequence is taken from Ref. 1. The dashed circles are residues of variable presence, involved in the amino-terminus heterogeneity. The asterisk ( * ) marks position - 8 , which is filled with either an Asp (D) or an Asn (N) residue.

proton) as shown by a selective decoupling experiment. A comparison with K1 and K4 (Fig. 6A,B) assigns this set of multiplets to the (conserved) Leu-46 residue. By analogy to other kringles, we can tentatively identify the remaining pair of methyl doublets at 0.4 ppm as arising from residue 77, which, in the case of K5, is Val-77 (Fig. 6A-C). There is a sharp singlet at 2.18 ppm (Fig. 5) whose position corresponds to that of a Met -CH~ group in a random-coil polypeptide. As the K5 fragment contains only one Met residue, we can assign this peak to Met-2. Fig. 7A shows the low-field spectrum of K5 dissolved in aH20. This spectral region contains both aromatic CH resonances as well as signals stemming from exchangeable, mostly peptidyl amide, His imidazole and Trp indole, N H groups. Fig. 7C depicts, as a reference, the same spectral region after the exchange-labile NH sites have been fully deuterated in 2H=O. For comparison, Fig. 7B shows a spectrum acquired continuously,

260

0 D ::I:

ppm Fig. 5. IH-NMR spectrum of human plasminogen kringle 5 at 611 MHz. The sample was dissolved in 2H20 and the spectrum was recorded after exchange of labile hydrogen atoms for deuterium. The asterisk (*) denotes the sharp signal from the internal dioxane reference. K5 concentration ---1 mM, pH* 4.5, 37°C. The spectrum is shown resolution-enhanced. Leu 46

Vo177

~

*

C

~-_--~___

Kringle

5

Leu 77 __

_

L e 046

Leu 46

Irerr

0

-i

- ~

ppm

Fig. 6. 1H-NMR spectra of homologous human plasminogen kringles at 300 MHz: high-field region. A, kringle 1; B, kringle 4; C, kringle 5. All spectra were recorded in 2H20 after exchange of labile hydrogen atoms for deuterium. The asterisks (*) indicate signals from aliphatic contaminants. For all spectra, kringle concentration is 1 raM, pH* 4.5, 37°C. The spectra are resolution-enhanced.

for 10 h, immediately after dissolving the sample in 2H20. It is clear, from a comparison of spectra A and B, that despite the relatively high temperature, 37 ° C, a number of N H groups exhibit retarded 1H-2H exchange kinetics. This feature, in combination with the chemical shift range of a number of peptidyl amide N H signals (10 > 3 > 7.8 ppm, Fig. 7A) indicates a globular conformation endowed with substantial intramolecular H-bonding and tertiary structure. Resonances which appear in t H 2 0 at 6 > 11.5 p p m are reminiscent of similar signals in the K4 spectrum, many of which arise from Trp side chain N H groups [40]. Such highly shifted resonances afford further evidence for a compact structure in which aromatic rings are densely packed against each other. Protein aromatic spectra often exhibit relatively narrow, apparently unsplit peaks stemming from either His imidazole H2 and H4 or the Trp indole H2 protons. K5 contains two histidine (sites 31 and 33) and two tryptophan (sites 25 and 62) residues; therefore six singlets are expected in the aromatic spectrum. Four of these, peaks 3-6, can be readily followed through an a c i d / b a s e titration (Fig. 8). Peaks 1 and 2 were identified by COSY

261

SOLVENT

ZH20 (fully exchonged )

2H20

B

(poriiolly exchanged )

.~_~_j"k.j

15

¸. j

12

i

~._.._~._~

11

1'0

°b

8

l~

7

'",

~

il ,

~' , '

~-,

1 H 20

6

ppm Fig. 7. 1H-NMR spectra of kringle 5 at 300 MHz: low-field region. A, 2.5 mM K5 in IH20, pH 4.5, 37°C; B and C, 1 mM K5 in 2H20 , pH* 4.5, 37°C. Spectrum A was recorded using the two hard-pulse technique [36]. Spectrum B was accumulated for 10 h after dissolving the protein in 2H20. Spectrum C was recorded after the protein had been fully exchanged with 2H20. Spectrum A is shown with slight resolution-enhancement.

and spin-echo experiments. As seen in Fig. 8, spectra A, B and C, recorded at pH* 8.7, 7.1 and 4.5, respectively, singlets 3, 4 and 5 are the most sensitive to acid/base perturbation. The singlets' chemical shift versus pH profile is shown in Fig. 9. Upon acidification, singlet 3 shifts approx. 1 ppm to low fields, while singlet 5 shifts only approx. 0.4 ppm in the same direction. Singlet 3 at 8.45 ppm and sing,let 5 at 8.41 ppm (Fig. 8c) titrate with p K * = 6.24 and PKa* = 6.28, respectively, and can therefore be identified as arising from the same His imidazole group (labelled H-II). Additionally, when incubated at 70 o C, singlet 3 disappears from the spectrum (an effect due to 1H-2H exchange against solvent

2H20); hence, it is identified as an imidazole H2 signal [37]. It follows that singlet 5 arises from the H4 on the same ring. At neutral pH, the relative order of these singlets on the chemical shift scale is opposite to what is observed for an exposed, random-coil His imidazole [37]. This 'reverse' ordering is also seen for His-31 in K1 and K4 [19,21,42] and, in conjunction with its pH* titration profile (Fig. 9), affords a criterion for assigning the His II peaks in K5 to the same, conserved residue. The other pair of His signals could be identified from a 300 MHz COSY experiment at pH* 4.5 (Fig. 10), as the two imidazole CH protons exhibit a small coupling which, depending on the

262

pH* Y-I

6

15

4

4.5

C "'I

,q

7.1

B

Y-I

6-'-

A

,' I0

c~

4' ..,j'%.d~J

"1 'hf

8

7

8.7 6

5

ppm Fig. 8. IH-NMR spectra of kringle 5 at 300 MHz: acid/base perturbation of aromatic resonances. The spectra were recorded in 2H20, 1 mM, 37°C, (A) p H * 8.7; (B) p H * 7.1: (C) p H * 4.5. His and Trip singlets are numbered according to their chemical shift ordering at p H * 7.1 (B). Spectra are shown with the same resolution enhancement.

line width, can often be detected as a COSY cross-peak [43]. Fig. l l B shows an absolute-mode COSY cross-section along 31 which unambiguously shows the very shifted singlet 6 at 9.65 p p m to be connected to singlet 1 at 7.26 ppm. Thus, the two should be paired as the H2 and H4 protons of the same His ring (labelled H-I). These singlets do not seem to shift significantly throughout the a c i d / b a s e titration (Figs. 8 and 9). Singlet 6 decreases in intensity at 70 ° C, indicating 1H_ 2H exchange with the deuterated solvent, and hence it

should be assigned to an imidazole H2 proton. It follows that singlet 1 must arise from the H4 on the same ring. This pattern and behavior matches the His-33 spin system of K4 [21,22]. It should be noted that in K5 the H2 resonance from this side chain is shifted approx. 1.2 p p m further to low field than is the analogous resonance in K4 [21]. There are two singlets which have not yet been assigned and which must come from the Trp residues. It has been shown that the Trp II (Trp-62) singlet in both K1 and K4 titrates slightly with

263

I

'

i

,

L

,

i

,

i

,

i

,

i

,

1

pK4* = 3.9 and = 4.8, respectively [19,21], possibly because the corresponding proton is near to an acidic side chain group. In K5, singlet 4 appears at approx. 7.89 ppm and titrates slightly with pK* = 4.95 (Figs. 8 and 9). By analogy to what is observed for the homologous domains, this suggests that in K5 singlet 4 is the indole H2 signal to Trp II (Trp-62). By elimination, singlet 2 at approx. 7.33 ppm must correspond to the aromatic H2 of the Trp I (Trp-25) indole spin system which is conserved in all kringles which have been sequenced [1,6-10]. By comparing K5 spectra recorded at pH* 7.1 and 4.5 (Fig. 8, B and C), it is clear that substantial line-narrowing occurs upon acidification while broadening becomes pronounced under alkaline conditions (spectra A, pH* 8.7) and is particularly noticeable for singlet 6, which stems from the His-33 imidazole H2 proton. Raising the pH* above 8.7 drastically deteriorates the quality of the spectrum and suggests incipient unfolding at pH* > 9. Thus, under neutral and alkaline conditions, K5 yields a substantially broader spectrum than either K1 or K4. It is possible that this is due to extensive kringle aggregation when in absence of ligand (as is thought to be the case for the homo-

,

9.

S (ppm)

•

8.C

H-I

@

W-I

L

2

I

h

3

[

,

i

4

,

5

i

L

i

h

I

6 7 pH ~

i

L

8

i

9

io

Fig. 9. Acid/base titration of aromatic singlets in the ~H-NMR spectrum of kringle 5. Data points are labelled according to the singlet numbering used in Fig. 8. The data were obtained from spectra recorded at 300 MHz, 37°C after exchange of labile hydrogen atoms for deuterium: n, His-I; i , His-II; open symbols, H4 resonances; filled symbols, H2 resonances; • denotes Trp H2 data. The derived p K * values are 6.26 for His-II (His-31), and 4.95 for Trp-II (Trp-62); His-I (His-33) and Trp-I (Trp-25) titrate only slightly.

J

I

W-I w-Tr

6

7

81 (ppm) D

8

9

H-I ''[

. . . .

I

9

. . . .

I

. . . .

I

8

. . . .

I

. . . .

t

7 82(ppm)

. . . .

1

. . . .

I

6

. . . .

~

. . . .

Fig. 10. Contour plot of the aromatic tHNMR COSY spectrum of kringle 5 at 300 MHz. Scalar conneetivities for the two Trp and two His side chains are indicated. The spectrum, recorded in absolute mode, is shown resolution-enhanced with a sine bell function in both dimensions. Two Trp crosspeaks, connecting broad indole triplets, are marked ®; they can be detected in spectra treated with Gaussian convolution. Kringle concentration ~-1 raM, pH* 4.5, 37°C.

264

2

H-It

Y-I

-.

ii

,ii !,

i

COSY cross-section

B ,

I0

<$

•

.

8

7

ppm Fig. 11. Identification of kfingle 5 histidyl and tryptophanyl IH-NMR singlets at 300 MHz. (A) Aromatic region of the one-dimensional spectrum of K5 (resolution enhanced). Singlets are numbered as indicated in Fig. 8. His-II (His-31) singlets are paired according to acid/base titration data (Figs. 8 and 9). Singlets 2 and 4 stem from Trp-1 (Trp-25) and Trp-II (Trp-62), respectively. (B) COSY spectrum cross-section: the intense peak at 9.6 ppm represents the diagonal H2 signal of His-II (His-33), while the smaller peak, at 7.22 ppm, is the cross-peak arising from the H4 proton, J-coupled to the H2 proton. The complete, aromatic COSY spectrum is shown in Fig, 10.

logues); the relatively longer N-terminal tail of K5 is likely to be involved in this interaction. It is thus tentatively concluded that acidic conditions are to be preferred for ]H-NMR spectral analysis of the K5 fragment. Fig. 10 shows the aromatic COSY spectrum of K5 recorded at pH* 4.5: connectivities among Trp indole cross-peaks are indicated. Two features are worth noticing: (a) the spread of indole resonances, ranging between = 5.15 and --- 8.25 ppm; (b) the analogy with the Trp-I (Trp-25) and Trp-II (Trp-62) spectra in K1 [19] and K4 [22,42]. Feature (a) suggests close interactions among aromatic groups which induce mutual anisotopic ring current shifts. Feature (b) further underscores the structural homology among the various plas-

minogen kringles. The labelling of the Trp I and Trp II aromatic spin systems (Fig. 10) corresponds to that of the singlets (Fig. 9) and is based on comparisons with the other kringles. The pairing of the Trp I and Trp II singlets to the rest of their indole spin systems has been unambiguously established in K1 and K4 [19,40,42]. Among the five plasminogen kringles, K1 affords the closest homologue to K5. Fig. 12 compares 300 MHz aromatic spectra of K1 (A) and K5 (B) at pH* 4.5, 37 o C. Consistent with Figs. 8, 9 and 11, side chain His and Trp singlets are numbered 1-6 in the K5 spectrum B. It is clear that there is an overall spectral analogy between the two kringles. The identification of selected Tip and Tyr multiplets is based on the COSY experi-

265 W-I

'7

2

H-I

H-IT

Y-I

N 5

L

W-~ 4

W-IT

-~-~--

il His3'L~ •

I0

_

9

'

Kringle 5

L

I

I__

~lHis 4~'

I' '

Kringle 1

Tyrr2

r

8

7 ppm

6

Fig. 12. 1H-NMR aromatic spectra of human plasminogen kringles 1 (A) and 5 (B) at 300 MHz. His and Trp singlets are numbered 1-6 in spectrum B, according to their chemical shift ordering at neutral pH (Fig, 8). One-letter amino acid code labels are used to mark K5 signals arising from conserved residues in the two homologues. The assignment of His-31, His-41 and Tyr-72, signals in K1 has been reported elsewhere [16]. The spectra are shown resolution-enhanced. An unidentified contaminant resonance, which overlaps with singlet 3 in spectrum B, is labelled with an asterisk ( * ). Kringle concentration -- 1 mM, pH * 4.5, 37 o C.

ment (Fig. 10) and on comparative analysis of K1, K4 and K5 spectra. In K1, Tyr I (Tyr-72) resonances are found to be high-field-shifted and provide a characteristic AA'BB' pattern at approx. 6.27 ppm; these signals shift further when ligand is present [14]. There is a corresponding pair of doublets in the K5 spectrum at approx. 6.27 ppm yielding a quasi-degenerate AA'A"A" pattern. Since Tyr-72 is conserved, we assign this pair of overlapping doublets to that residue (also labelled Y-I in Figs. 8 and 11). The overall similarity of the Tyr I (Tyr-72), Tyr II (Tyr-50), Trp I (Trp-25), Trp II (Trp-62) and His II (His-31) spectra in K1 and K5 strongly suggests that the environment around these residues is very much the same in the two kringles. Indeed, in combination with the Leu-46 CH83"~" doublets at 0.38 and -1.04 ppm (Fig. 6), the characteristic Tyr I aromatic spectrum provided an independent criterion to monitor the

presence of the K5 fragment in the course of its isolation/purification.

Benzamidine binding to kringle 5 The ligand specificity of K5 has been shown to be different from that of K1 and K4 [26,27]. Fig. 13 shows the effect of benzamidine-binding on the 611 MHz spectrum of K5 at pH* 7.2. Ligand-binding sharpens the protein spectrum significantly, as noticed by comparing spectra A and C. As is the case with other kringles [14,20], we attribute this effect to ligand interference with self-aggregation. Ligand-induced shifts are apparent within the group of Tyr resonances at approx. 6.95 ppm and can be readily followed for the three isolated, shifted multiplets labelled a, b and c in spectrum C. Multiplets a and c arise from Trp II (Trp-62) and b from Tyr I (Tyr-72), suggesting, as is the case with K1 [14] and K4 [15,16], that residues at

266

[Ligond]/[K 5 ] HzN~-j,N H 2

H4

Benzamddine

S

D

CO (free hgond)

H2',6 R3,5 H4

I I

C

6.23

I

2.9 5

B

I

............ ,',j,~,v,'

A 10

9

8

7

6

O. 0 0

5

ppm

Fig. 13. Effect of benzamidine-binding on the aromatic tH-NMR spectrum of kringle 5 at 600 MHz. (A) Ligand-free K5; (B) K5 in the presence of approx. 3-fold excess benzamidine; (C) K5 in the presence of approx. 6-fold excess berlzamidine; and (D) free benzamidine. The vertical connections denote protein and ligand resonances' shifts. Spining side bands arising from the ligand are indicated (s). Peaks specifically monitored for ligand-binding effects are labelled a, b, c in spectrum C (titration curves are shown in Fig. 14). The molecular structure of benzamidine is shown as an inset to spectrum D. Assigned ligand resonances are indicated: H4 (one-proton triplet), H2,6 (two-proton doublet) and H3,5 (two-proton triplet). The spectra are shown resolution-enhanced. Kringle concentration ~ 1 raM, pH* 7.2, 37°C.

these two sites are involved in l i g a n d - b i n d i n g . I n contrast, n o significant shifts are observed in the aliphatic spectrum. High static magnetic fields help to visualize the ligand spectrum b y simplifying it to a first-order pattern. The 611 M H z spectrum of free b e n z a m i dine shows two groups of multiplets at 7.60 a n d 7.75 p p m (Fig. 13D). I n t e g r a t i n g these multiplets

shows that the area ratio for the l o w - f i e l d / h i g h field resonances is 3 : 2. The high-field multiplet at 7.60 p p m is a triplet a n d thus represents the t w o - p r o t o n ligand H3,5 signal. I n s p e c t i o n of the low-field resonances at 7.75 p p m indicates that a o n e - p r o t o n triplet (H4) overlaps with a two-proton d o u b l e t (H2,6). Analysis of spectra of the protein-ligand com-

267

plex (Fig. 13, B and C) shows that upon binding to the kringle the ligand H2,6 doublet moves the most, the H4 triplet moves the least, and the H3,5 triplet experiences a shift of intermediate magnitude. At [K5]/[benzamidine] ~ 1 : 3, the ligand H3,5 triplet and H2,6 doublet appear at approximately the same frequency, causing the multiplicity of these peaks due to mutual coupling to be lost (Fig. 13B). Furthermore, at this ligand level, the benzamidine H4 triplet, is appreciably broadened, most likely because of on-off exchange. This suggests that the amidino (cationic) end of the benzamidine molecule is more intimately involved in the ligand-kringle interaction. This is of possible functional relevance for K5, since arginine contains an analogous guanidino group and has been shown by affinity chromatography studies to elute miniplasminogen [27]. Fig. 14 shows the chemical shift ligand titration profile of the kringle 5 peaks labelled a, b and c in Fig. 13. The data for resonances a and b have been fitted to a single-site binding equation (Langmuir adsorption isotherm) as described [14,39] to yield a K a = 1.7 + 0.4 mM -1 at pH* 7.2, 37 ° C It is clear from the non-saturation trend exhibited by curve c that other secondary binding

20-

o-- 0 b

181614-

•( p p m x l O

z)tO_

C

86420 0

'

'

'

' a'~ . . . .

I10 . . . .

ii 5 ,

,

,

r 210 ,

,

,

[So]/[Ko] Fig. 14. Benzamidine titration of kringle 5. Data points extracted from 1 H - N M R experiments at 300 M H z represent shifts induced on multiplets a, b and c (Fig. 13) upon ligandbinding. The graph plots AS, the chemical shifts of K5 resonances in the presence of |igand referred to those in the free kringle, versus the concentraton ratio of total ligand to total kringle [So]/[Ko].

site(s) are present. Relative to ligand-binding to other kringles, the benzamidine/K5 interaction is rather weak: K a = 145 mM -1 and ----74 mM -1 have been measured for binding of p-benzylaminesulfonic acid (BASA), an aromatic ligand, to K1 and K4, respectively, at pH* 7.2, 25°C [14,20,39]. Both the lower affinity and different specificity of K5 might reflect the absence, in this kringle, of the ion pair interaction mediated by Arg-71 and, possibly, Arg-34 in the homologues [44,45]. At any rate, we have established that the plasminogen fragment we have isolated satisfies the criterion of benzamidine-binding to be expected from a K5-containing species [26]. Discussion

The scheme for purification of K5 we now report has several advantages over previously published methods. First, the pepsin cleavage site precedes the interchain disulfide bonds so that cystine reduction and alkylation are not necessary. Second, the K5 product of this digestion carries a relatively short C-terminal non-kringle ' tail'. When compared to the K5 which is isolated with the method of Ref. 18, 15 amino acids are eliminated. This decreases the complexity of the aliphatic 1H-NMR spectrum and is likely to lower the extent of kringle self-aggregation. A third advantage of our purification scheme is that, unlike the method of Novokhatny and coworkers [5], it avoids HPLC purification procedures. Indeed, by resorting to conventional column chromatography, the larger amounts of K5 needed for N M R studies can be readily obtained. Furthermore, while the original paper [5] does not specify the composition of the K5 fragment, we have been able to monitor the purification steps and characterize the final product in terms of both amino acid analyses and 1H-NMR spectroscopy. The latter provided a most valuable tool for monitoring progress through the purification procedure. This method has been repeated over 15 times and typically yields approx. 20-30 mg of K5 fragment per gram of miniplasminogen starting material. We present the first N M R study of human plasminogen K5. A preliminary spectroscopic characterization indicates a compact, globular structure for K5. We have fully identified and

268

assigned the imidazole resonances of the two histidine side chains, the indole subspectra of the two tryptophan residues, and the aromatic signals from Tyr-72. In the aliphatic spectrum we identify the singlet due to Met-2, the CH~ "g doublets of Leu-46 and (tentatively) the CH~ ,v resonances of Val-77. Overall, the spectum of K5 shows close similarity to those reported for K1 [14] and K4 [20-24]; this points to a global structural homology among the three plasminogen kringles. This is underscored by two characteristic sets of shifted signals present in all three kringles' spectra: (a) the two high-field doublets at 0.38 and -1.04 ppm, assigned to Leu-46 (Fig. 6), and (b) the resonance at 11.8 ppm due to an exchange-retarded NH proton (Fig. 7B). This second resonance is also found in prothrombin kringles [41] and has been assigned, in the plasminogen kringles, to the Trp I (Trp-25) indole NH proton [40,42]. In contrast, the unusual chemical shift of the His CH2 singlet at 9.6 ppm is unique to K5 (Figs. 5 and 7). It is interesting to note the large shift of the Trp II (Trp-62) indole doublet at 5.97 ppm in K5 relative to its position in K1 at 6.55 ppm (Fig. 12). In conjunction with the singlet at 9.65 ppm, this strongly suggests that the environment around some of the aromatic residues in K5 is somewhat different from that encountered in either K1 or K4 [19,42]. This may be a consequence of the additional residue at position 35 in K5 (a deletion in K1 and K4), which could impose additional constraints on the packing of side chains within the structure. We verify that our isolated fragment binds benzamidine, adding evidence that we have indeed obtained K5 [26]. The NMR data suggest that, overall, the structure of the K5 benzamidine-binding site is likely to be closely similar to that of the homologous plasminogen K1 and K4 lysine-binding sites. The relatively lower ligand affinity of K5 is probably a consequence of this kringle-ligand interaction missing the added stability conferred by the ion pairing through the Arg-71 side chain, present in the homologous domains but absent in K5. However, the fifth kringle carries Asp-55 to interact with the cationic end of the ligand [44,46], which might contribute to its specificity toward benzamidine.

Acknowledgements The authors would like to express their gratitude to Dr. William E. Brown of Carnegie Mellon University for his valuable help with the amino acid composition and sequence analyses. This research was supported by the U.S. Public Health Service, NIH grant HL 29409. The 600 MHz N M R facility is supported by grant RR 00292 from the National Institutes of Health.

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34 Clore, G.M., Kimber, B.J. and Gronenbom, A.M. (1983) J. Magn. Resonance 54, 170-173 35 Ernst, R.R. (1966) Adv. Magn. Resonance 2, 1-135 36 Ferrige, A.G. and Lindon, J.C. (1978) Magn. Resonance 31, 337-340 37 Markley, J.L. (1975) Acc. Chem. Res. 8, 70-80 38 Wider, A., Macura, S, Kumar, A., Ernst, R.R. and Wi~thrich, K. (1984) J. Magn. Resonance 56, 207-234 39 De Marco, A., Petros, A.M., Laursen, R.A. and Llinfis, M. (1987) Eur. Biophys. J., in the press 40 Motta, A., Laursen, R.A. and Llinfis, M. (1986) Biochemistry 25, 7924-7931 41 Esnouf, M.P., Israel, E.A., Pluck, N.D. and Williams, R.J.P. (1980) in The Regulation of Coagulation (Mann, K.G. and Taylor, F.F., eds.), pp. 67-74, Elsevier/North-Holland, New York 42 De Marco, A., Pluck, N.D., Bfinyai, L., Trexler, M., Laursen, R.A., Patthy, L., Llinfis, M. and Williams, R.J.P. (1985) Biochemistry 24, 748-753 43 King, G. and Wright, P.E. (1982) Biochem. Biophys. Res. Commun. 106, 559-565 44 Trexler, M., Villi, Z. and Patthy, L. (1982) J. Biol. Chem. 257, 7401-7406 45 Villi, Z. and Patthy, L. (1984) J. Biol. Chem. 259, 13690-13694 46 Lerch, P.G. and Rickli, E.E. (1980) Biochim. Biophys. Acta 625, 374-378