Proton Overhauser experiments on kringle 4 from human plasminogen. Implications for the structure of the kringles' hydrophobic core

1H-NMR Overhauser experiments at 300 and 600 MHz have been implemented on the isolated kringle 4 fragment of human plasminogen. This study shows that Leu46 and Leu77 CH3 delta,delta' groups, as well as two threonine CH3 gamma and a methionine S-CH epsilon (probably Met48) groups, are in efficient dipolar contact with histidine and aromatic side-chains. In particular, the experiments reveal that of the two Leu46 CH3 delta,delta' groups, one is in efficient contact with tryptophan (Trp25 and Trp62) indole rings while the other interacts with a tyrosine (probably Tyr41) phenol. Leu46 appears also to be close to an Ala CH3 beta group. Such a hydrophobic cluster appears to be contiguous to Trp72, hence to Arg71, residues that are through to be part of the lysine-binding site. Acid-base titration experiments show that the buried methionine S-CH3 epsilon group senses a neighboring ionizable group of pK*1 = 3.76, suggesting presence of a carboxyl anionic group (probably an aspartic acid side-chain) in the vicinity of the hydrophobic core. A preliminary model is proposed for the overall folding of the kringle polypeptide chain.     

Analysis of the aromatic 1H-NMR spectrum of the kringle 5 domain from human plasminogen. Evidence for a conserved kringle fold

A kringle 5 domain fragment from human plasminogen has been investigated by 1H-NMR spectroscopy at 300 MHz and 620 MHz. The study focuses on the kringle 5 aromatic spectrum as aromatic side chains appear to mediate the binding of benzamidine. Spin-echo experiments and acid/base-titration studies in conjunction with two-dimensional double-quantum and chemical-shift-correlated spectroscopies were used to identify individual spin systems. Sequence-specific assignments of aromatic resonances are derived from direct comparison of the kringle 5 spectrum with spectra of the homologous kringle 1 and kringle 4 domains of plasminogen. As previously observed for kringles 1 and 4, the pattern we detect for Tyr9 in kringle 5 reflects a slow conformational exchange between two states in equilibrium, one in which the Tyr9 ring is freely mobile and one in which its flip dynamics are constrained. Proton Overhauser experiments in 1H2O and in 2H2O have been used to probe aromatic ring interactions and to identify residues which are part of the hydrophobic core centered at the Leu46 side chain. Overall, the data indicate a strong structural homology among the three plasminogen kringles.     

The aromatic 1H-NMR spectrum of plasminogen kringle 4. A comparative study of human, porcine and bovine homologs

The isolated kringle 4 domain of human plasminogen has been compared with homologous structures from bovine and porcine sources, both free and in the presence of the ligand 6-aminohexanoic acid, by two-dimensional 1H-NMR spectroscopies at 300 MHz and 600 MHz. The chemical-shift-correlated, spin-echo-correlated, and double-quantum-correlated aromatic spectra of the three proteins reveal that the globular conformation of the fourth kringle is closely maintained throughout the set of homologs. Direct comparison shows that the three conserved Trp residues (at sites 25, 62 and 72) which exhibit highly non-degenerate subspectra, find themselves in similar intramolecular environments. In particular, proton Overhauser experiments reveal that the close steric interaction between the Trp-II (Trp62 or Trp25) indole group and the aromatic ring at site 74 (Tyr74 or Phe74) is strictly preserved. This feature forces the kringle inner loop, closed by the Cys51-Cys75 link, to fold back onto itself so as to place the site 74 residue proximal to the Cys22-Cys63 bridge. Single-residue substitutions enable unambiguous assignments of His-I to His3, Tyr-III to Tyr41 and Tyr-IV to Tyr74. From this direct evidence, comparison with the kringle 1 spectrum, and the previously reported chemical modification of Tyr-II (Tyr50) [Trexler M., Bányai L., Patthy L., Pluck N. D. & Williams R. J. P. (1985) Eur. J. Biochem. 152, 439-446], Tyr-I and Tyr-V (the latter, an immobile ring on the 600-MHz time scale) could be assigned to Tyr2 and Tyr9, respectively. Since Trp-III has previously been assigned to Trp72 at the lysine-binding site, the present study completes the assignment of 10 out of 12 aromatic spin systems in the kringle 4 1H-NMR spectrum; the only ambiguity which remains concerns the Trp-I and Trp-II indole spin systems, which are totally identified but as yet only tentatively assigned to Trp25 and Trp62, respectively.     

A 1H-NMR study of isolated domains from human plasminogen. Structural homology between kringles 1 and 4

Kringles 1 and 4 from human plasminogen are polypeptide domains of Mr approximately equal to 10000 each of which can be isolated by proteolysis of the zymogen. They have been studied by 1H-NMR spectroscopy at 300 MHz and 600 MHz. The spectra, characteristic of globular structures, show striking analogies that point to a close conformational relatedness among the two kringles, consistent with their high degree of amino acid conservancy and homology. The interaction of both kringles with p-benzylaminesulfonic acid (BASA), an antifibrinolytic drug that binds to a lysine-binding site, results in better resolved, narrower lines for both spectra. Aromatic and methyl-region spectra of BASA complexes of kringles 1 and 4 were compared and the latter was studied by two-dimensional NMR spectroscopy. Analysis of the CH3 multiplets in terms of their resonance patterns, and the amino acid compositions and sequences of the two kringles, leads to the identification of most signals and to some assignments. In particular, a doublet at -1 ppm, exhibited by both kringles and also found in reported proton spectra of homologous bovine prothrombin fragments, has been assigned to Leu46, a residue that is conserved in all of the kringles studied to date by 1H-NMR. Since this resonance is somewhat more sensitive to BASA than other methyl signals, it is likely that Leu46 is proximal to the lysine-binding site. Nuclear Overhauser experiments reveal that Leu46 is surrounded by a cluster of closely interacting hydrophobic and aromatic side chains. Kringle 4 was also compared with a derivative chemically modified at Trp72 with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide. As judged from the proton spectra, the modified kringle 4 retains globularity and is perturbed mainly in the aromatic region, in analogy to that which is observed for the unmodified kringle upon BASA binding. Furthermore, although previous studies have indicated no retention of the modified kringle by lysine-Sepharose, the NMR studies point to a definite interaction between BASA and the kringle derivative. The spectroscopic data also suggest that the His31 imidazole is not significantly affected by the ligand and that the lysine-binding site is structured mostly by hydrophobic side chains, including Trp72 in the case of kringle 4, and probably Tyr72 in kringle 1.     

Proton magnetic resonance study of lysine-binding to the kringle 4 domain of human plasminogen. The structure of the binding site

The binding of L-Lys, D-Lys and epsilon-aminocaproic acid (epsilon ACA) to the kringle 4 domain of human plasminogen has been investigated via one and two-dimensional 1H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Ligand-kringle association constants (Ka) were determined assuming single site binding. At 295 K, pH 7.2, D-Lys binds to kringle 4 much more weakly (Ka = 1.2 mM-1) than does L-Lys (Ka = 24.4 mM-1). L-Lys binding to kringle 4 causes the appearance of ring current-shifted high-field resonances within the -1 approximately less than delta approximately less than 0 parts per million range. The ligand origin of these signals has been confirmed by examining the spectra of kringle 4 titrated with deuterated L-Lys. A systematic analysis of ligand-induced shifts on the aromatic resonances of kringle 4 has been carried out on the basis of 300 MHz two-dimensional chemical shift correlated (COSY) and double quantum correlated spectroscopies. Significant differences in the effect of L-Lys and D-Lys binding to kringle 4 have been observed in the aromatic COSY spectrum. In particular, the His31 H4 and Trp72 H2 singlets and the Phe64 multiplets appear to be the most sensitive to the particular enantiomers, indicating that these residues are in proximity to the ligand C alpha center. In contrast, the rest of the indole spectrum of Trp72 and the aromatic resonances of Trp62 and Tyr74, which are affected by ligand presence, are insensitive to the optical nature of the ligand isomer. These results, together with two-dimensional proton Overhauser studies and ligand-kringle saturation transfer experiments reported previously, enabled us to generate a model of the kringle 4 ligand-binding site from the crystallographic co-ordinates of the prothrombin kringle 1. The latter, although lacking recognizable lysine-binding capability, is otherwise structurally homologous to the plasminogen kringles.     

Ligand interactions with the kringle 5 domain of plasminogen. A study by 1H NMR spectroscopy

The binding of small molecules to the kringle 5 domain fragment of human plasminogen has been investigated by 1H NMR spectroscopy at 300 MHz. The compounds tested as potential ligands include L-arginine, L-lysine, and a number of aliphatic and aromatic analogs of similar size but different ionic charge configurations. Ligand/kringle 5 association constant (Ka) values were obtained from ligand titration experiments at 22 degrees C, pH 7.2. Neither L-arginine nor N alpha-acetyl-L-arginine and N alpha-acetyl-L-arginine methyl ester bind measurably to kringle 5 (Ka approximately less than 0.05 mM-1). In contrast, binding of hexylamine or epsilon-aminocaproic acid (epsilon ACA) is favored (Ka approximately 2.9 and 10.5 mM-1, respectively). Benzamidine and p-benzylaminesulfonic acid associate with kringle 5 with similar affinities (Ka approximately 3.4 and 2.2 mM-1, respectively) while benzylamine binds about twice as tightly (Ka approximately 6.3 mM-1). The higher affinities toward both benzylamine and epsilon ACA indicate that a free carboxylate group is not, by itself, a main determinant of ligand-binding to kringle 5. The experiments also reveal a definite affinity for L-arginine methyl ester, L-lysine, and N alpha-acetyl-L-lysine methyl ester. It is suggested that, although weak (0.1 approximately less than Ka approximately less than 0.6 mM-1), these interactions could be of physiological relevance in the context of plasminogen binding to the fibrin clot. Ligand-induced shifts of kringle 5 proton resonances indicate that the Trp25, His33, Tyr50, Trp62, and Tyr72 (kringle numbering convention) side chains form or neighbor the kringle 5-binding site. Benzamidine-kringle 5 magnetization transfer (Overhauser) experiments verify a close proximity of the bound ligand to these aromatic groups. A model of the binding site is proposed in which the above residues interact closely with each other and define a lipophilic surface which is accessible to the free ligand.     

The binding of plasminogen fragments to cultured human umbilical vein endothelial cells.

Glu-plasminogen, kringle 1-5, kringle 1-3, and miniplasminogen exhibited strong binding to human umbilical vein endothelial cells (HUVEC). On the other hand, no significant binding was obtained with microplasminogen and kringle 4. Kringle 1-5 and miniplasminogen, which both contained kringle 5, specifically inhibited the binding of plasminogen to HUVEC while kringle 1-3 did not. The results implied plasminogen molecule contained at least two binding sites, with which it interacted HUVEC. The stronger binding site was located in kringle 5 and the weaker one was in kringle 1-3. Kringle 4 and the active site domain exhibited no significant binding to HUVEC. The interaction of plasminogen with HUVEC is mainly through binding site on kringle 5.     

Analysis of the aliphatic 1H-NMR spectrum of plasminogen kringle 4. A comparative study of human, porcine, bovine and chicken homologs

The aliphatic 1H-NMR spectrum of the kringle 4 domain of human plasminogen has been studied via two-dimensional chemical shift correlated (COSY) and nuclear Overhauser correlated (NOESY) experiments at 300 MHz and 620 MHz. A number of aliphatic proton spin systems have been identified and several definite assignments have been made. This was mainly achieved by comparison of the human kringle 4 spectrum with spectra of the porcine, bovine and chicken homologs and also with that of the kringle 1 from human plasminogen on which we have reported previously. The three valyl and two leucyl residues of human kringle 4 have been assigned. The eleven threonyl spin systems have been identified via a RELAYED-COSY experiment and Thr17 has been assigned. The three alanyl spin systems have been identified and assigned. Six seryl spin systems have been identified and the signals from the seven glycyl residues of human kringle 4 have been located with Gly45 assigned. Furthermore, 24 AMX spin systems have been mapped in the COSY spectrum of human kringle 4 and H alpha-H beta,beta' spin systems of Tyr2, Tyr41, Tyr50, Tyr74, Trp25 and Trp62 have been assigned. From the spectrum of a deglycosylated chicken homolog, the epsilon-methyl singlets of Met28 and Met48 have been assigned. Finally, ligand effects on selected aliphatic resonances were observed which could be analyzed in terms of residues likely to neighbor the kringle lysine-binding site.     

Apolipoprotein(a): structure-function relationship at the lysine-binding site and plasminogen activator cleavage site

Apolipoprotein(a) [apo(a)] is the distinctive glycoprotein of lipoprotein Lp(a), which is disulfide linked to the apo B100 of a low density lipoprotein particle. Apo(a) possesses a high degree of sequence homology with plasminogen, the precursor of plasmin, a fibrinolytic and pericellular proteolytic enzyme. Apo(a) exists in several isoforms defined by a variable number of copies of plasminogen-like kringle 4 and single copies of kringle 5, and the protease region including the backbone positions for the catalytic triad (Ser, His, Asp). A lysine-binding site that is similar to that of plasminogen kringle 4 is present in apo(a) kringle IV type 10. These kringle motifs share some amino acid residues (Asp55, Asp57, Phe64, Tyr62, Trp72, Arg71) that are key components of their lysine-binding site. The spatial conformation and the function of this site in plasminogen kringle 4 and in apo(a) kringle IV-10 seem to be identical as indicated by (i) the ability of apo(a) to compete with plasminogen for binding to fibrin, and (ii) the neutralisation of the lysine-binding function of these kringles by a monoclonal antibody that recognises key components of the lysine-binding site. In contrast, the lysine-binding site of plasminogen kringle 1 contains a Tyr residue at positions 64 and 72 and is not recognised by this antibody. Plasminogen bound to fibrin is specifically recognised and cleaved by the tissue-type plasminogen activator at Arg561-Val562, and is thereby transformed into plasmin. A Ser-Ile substitution at the activation cleavage site is present in apo(a). Reinstallation of the Arg-Val peptide bond does not ensure cleavage of apo(a) by plasminogen activators. These data suggest that the stringent specificity of tissue-type plasminogen activator for plasminogen requires molecular interactions with structures located remotely from the activation disulfide loop. These structures ensure second site interactions that are most probably absent in apo(a).     

Kringle-2 domain of the tissue-type plasminogen activator. 1H-NMR assignments and secondary structure

A recombinant 90-residue polypeptide fragment containing the three-loop kringle-2 domain of human tissue-type plasminogen activator (t-PA) has been studied by two-dimensional 1H-NMR spectroscopy at 500 MHz. Complete sequence-specific resonance assignments were derived. Overall, the kringle exhibits a compact, folded conformation with more than 50% of the residues in irregular structures. Elements of secondary structure were identified from sequential, medium- and long-range dipolar (Overhauser) interproton interactions. These identifications were corroborated by analysis of spin-spin scalar 3J alpha N splittings and identification of backbone amide NH protons exhibiting retarded 1H/2H exchange in 2H2O. Three antiparallel beta-sheets and six tight turns were located. In addition, one short alpha-helical region was found in the Ser43-Ala44-Gln44a-Ala44b-Leu44c-Gly45+ ++ segment; this region contains three-residue insertions unique to the t-PA and urokinase kringles. Although the secondary structure of the t-PA kringle 2 in solution is in overall agreement with that observed in the crystallographic structure of the prothrombin kringle 1 [Tulinsky, A., Park, C.H. & Skrzypczak-Jankun, E. (1988) J. Mol. Biol. 202, 885-901], the alpha-helical segment and other details of the secondary structure differ somewhat from the prothrombin homolog.     

High-resolution crystal structure of apolipoprotein(a) kringle IV type 7: insights into ligand binding

Apolipoprotein(a) [apo(a)] consists of a series of tandemly repeated modules known as kringles that are commonly found in many proteins involved in the fibrinolytic and coagulation cascades, such as plasminogen and thrombin, respectively. Specifically, apo(a) contains multiple tandem repeats of domains similar to plasminogen kringle IV (designated as KIV(1) to KIV(10)) followed by sequences similar to the kringle V and protease domains of plasminogen. The KIV domains of apo(a) differ with respect to their ability to bind lysine or lysine analogs. KIV(10) represents the high-affinity lysine-binding site (LBS) of apo(a); a weak LBS is predicted in each of KIV(5)-KIV(8) and has been directly demonstrated in KIV(7). The present study describes the first crystal structure of apo(a) KIV(7), refined to a resolution of 1.45 A, representing the highest resolution for a kringle structure determined to date. A critical substitution of Tyr-62 in KIV(7) for the corresponding Phe-62 residue in KIV(10), in conjunction with the presence of Arg-35 in KIV(7), results in the formation of a unique network of hydrogen bonds and electrostatic interactions between key LBS residues (Arg-35, Tyr-62, Asp-54) and a peripheral tyrosine residue (Tyr-40). These interactions restrain the flexibility of key LBS residues (Arg-35, Asp-54) and, in turn, reduce their adaptability in accommodating lysine and its analogs. Steric hindrance involving Tyr-62, as well as the elimination of critical ligand-stabilizing interactions within the LBS are also consequences of this interaction network. Thus, these subtle yet critical structural features are responsible for the weak lysine-binding affinity exhibited by KIV(7) relative to that of KIV(10).     

Analysis of the aromatic 1H NMR spectrum of chicken plasminogen kringle 4

The intact kringle 4 domain of chicken plasminogen has been characterized by 1H NMR spectroscopy at 300 and 620 MHz in both the presence and absence of epsilon-aminocaproic acid, an antifibrinolytic drug. The study focuses on the aromatic resonances. Comparisons with spectra from human, porcine and bovine kringle 4 homologs indicates a strict conservancy of conformation, reflecting the underlying primary sequence homology, and leads to an unambiguous assignment of all the aromatic resonances, including those of Phe15 and His40 which are unique to the chicken domain. Conclusive evidence is found that the Tyr9 ring fluctuates between two states, one in which it flips fast and other in which it is severely hindered. Similarly, the Tyr64 side chain finds itself in a structurally constrained locus. The Trp62, Tyr64, and Trp72 aromatic resonances are most sensitive to ligand presence, supporting a previously reported model of the kringle 4 lysine-binding site. His40, Phe41, and Tyr74 are also perturbed by ligand indicating proximity to the site. In contrast, the Phe15 aromatic spectrum indicates a rather mobile phenyl ring which is insensitive to ligand presence, thus confirming the lesser importance of the corresponding segment within the first kringle loop in determining kringle structure and/or function.     

Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator.

A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ([K1tPA] residues C92-C173). The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ([K1HPg]) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which [K1tPA] was inserted. The recombinant (r) [K1HPg]PfXa[K1tPA] fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography. After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-[K1tPA]-containing polypeptide passed unretarded through the column. A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography. The purified r-[K1tPA], which contained the amino acid sequence SNAS[K1tPA]S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence. The molecular mass of r-SNAS[K1tPA]S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65). 1H-NMR spectroscopy and thermal stability studies of r-SNAS[K1tPA]S revealed that the purified material was properly folded and similar to other isolated kringle domains. Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-[K1tPA] domain occurred.     

1H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator

The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via 1H NMR spectroscopy at 300 and 620 MHz. The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli [Cleary et al. (1989) Biochemistry 28, 1884-1891]. The spectrum of t-PA kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4. Spectral comparison with human and bovine PGN kringle 4 identifies side-chain resonances from Leu46, which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems. Assignment of signals arising from the His13, His48a, and His64 side chains, which are unique to t-PA kringle 2, was assisted by the availability of a His64----Tyr mutant. Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant Ka approximately 11.9 mM-1. The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2. These include Tyr36 and, within the kringle inner loop, Trp62, His64, Trp72, and Tyr74. Acid/base titration of aromatic singlets in the presence of L-lysine yields pKa* approximately 6.25 and approximately 4.41 for His13 and His64, respectively, and shows that the His48a imidazole group does not protonate down to pH* approximately 4.3. Thus, the His48a and His64 side chains are in solvent-shielded locations. As observed for the PGN kringles, the Trp62 indole group titrates with pKa* approximately 4.60, which indicates proximity of the side chain to a titratable carboxyl group, most likely that of Asp57 at the binding site. Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2H2O for full deuteration in the presence of L-lysine at 37 degrees C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in 1H2O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His48a imidazole NH3 proton and the three Trp indole NH1 protons. A strong dipolar interaction was observed among the Trp25 indole NH1, the Tyr50 amide NH, and the His48a imidazole CH2 protons, which affords evidence for an aromatic cluster in t-PA kringle 2 similar to that found at the hydrophobic kernel of PGN kringles.(ABSTRACT TRUNCATED AT 400 WORDS)     

1H NMR studies of aliphatic ligand binding to human plasminogen kringle 4

A detailed 1H NMR analysis of ligand binding to the human plasminogen kringle 4 domain has been carried out at 300 MHz. The ligands that were investigated are N alpha-acetyl-L-lysine, L-lysine methyl ester, N alpha-acetyl-L-lysine methyl ester, L-lysine hydroxamic acid, trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA), and 4-(aminomethyl)bicyclo[2.2.2]octane-1-carboxylic acid (AMBOC). Specific ligand-binding effects were detected via two-dimensional COSY experiments. The side chains that are the most perturbed by ligand presence are those from Trp62, Phe64, and Trp72. Ligand-kringle saturation transfer (Overhauser) experiments show that the aromatic rings from these three residues, especially Trp72, are in direct contact with the ligand. These results add support to a previously reported model of the kringle 4 lysine-binding site [Ramesh, V., Petros, A. M., Llinás, M., Tulinsky, A., & Park, C. H. (1987) J. Mol. Biol. 198, 481-498] by which these aromatic groups are assigned a key role in establishing hydrophobic interactions with the ligand molecule. Equilibrium association constants (Ka) and kinetic rate constants (kon, koff) were determined for the binding of the various linear and cyclic ligands to kringle 4. We find that those ligands whose carboxylate function is blocked bind significantly weaker (Ka approximately less than 2 mM-1) than the corresponding analogues where the anionic center is present (Ka approximately greater than 20 mM-1), which underscores the relevance of the polar group in stabilizing the interaction with the kringle 4 binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

The X-ray crystal structure of full-length human plasminogen

Plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change.     

1H-NMR spectroscopic manifestations of ligand binding to the kringle 4 domain of human plasminogen

Structural aspects of the binding of the linear ligands N alpha-acetyl-L-lysine (AcLys) and epsilon-aminocaproic acid (epsilon ACA) and of the cyclic analogs trans-(aminomethyl)-cyclohexanecarboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) to the intact plasminogen kringle 4 domain have been investigated by 1H-NMR spectroscopy at 300 and 600 MHz. Ligand binding results in consistent shifts of the His-II (His31), Trp-I (Trp25?), Trp-II (Trp62?), Trp-III (Trp72), Tyr-II (Tyr50), and Phe64 ring signals. BASA tends to induce larger shifts than elicited by the aliphatic ligands, most noticeably on Trp-II and on Trp72, suggesting that the ligand aromatic ring interacts with the two indole groups. Trp-II and, to lesser extent, Trp-I interact with an acidic side chain group, in a manner that is blocked by BASA. BASA binding also perturbs Tyr-II (Tyr50), Tyr-III (Tyr41), and Tyr-IV (Tyr74) over a wide pH range and lowers the pKa* of His31 from approximately 4.8 to approximately 4.6. His-III (His33) responds to BASA and AMCHA but is relatively insensitive to the linear ligands. His33 carries a sterically shielded side chain which, in conjunction with Leu46, Trp-I, Tyr50, and Tyr74, participates in structuring the kringle hydrophobic core, contiguous to the binding site. Pronounced shifts are observed for aliphatic resonances stemming from the kringle-bound molecules of AMCHA, AcLys, and epsilon ACA. It is proposed that the lysine-binding site is mostly supported by the loop that extends from Cys51 through Cys71 and that aromatic residues, which include Trp-II, Trp72, and Phe64, play a major role in interacting with the nonpolar segment of the ligand molecule. The binding site also encompasses Tyr50, Tyr74, His31, and His33 although it is not clear the extent to which these residues interact directly with the ligand.     

600 MHz H nuclear magnetic resonance studies of the kringle 4 fragment of human plasminogen

Kringle 4, a approximately 10,000-Da domain in the heavy chain of human plasminogen, has been isolated intact and studied by H NMR spectroscopy at 600 MHz. The spectroscopic data indicates that kringle 4 possesses a globular and flexible structure which exhibits relatively fast amide-hydrogen exchange. About 17 NH groups show retarded exchange, with half-lives of approximately 7 h in 2H2O at pH* 6.45, 25 degrees C, which indicates that regions of the kringle are buried and shielded from direct interaction with the solvent. Analysis of the methyl region spectrum accounts for all singlets and doublets in terms of the amino acid composition; resonances from the C- and N-termini residues could be identified from the magnitude of their J couplings and their response to pH titration. It is shown that elastase digestion of plasminogen generates two species of kringle 4, one that terminates with Ala85 and another that extends to Val87. The heterogeneity can be resolved by chromatography on CM-Sephadex. The interaction of kringle 4 with BASA (p-benzylaminesulfonic acid), an antifibrinolytic drug presumed to bind to the plasminogen lysine-binding sites, has been investigated through the effects of added ligand on the kringle spectrum. The kringle lysine-binding site would appear to be integrated by a cluster of interacting His and aromatic residues since many of these resonances follow a definite saturation curve pattern upon BASA titration. In contrast, only minor changes are detected in the aliphatic methyl spectra. The association constant for the BASA-kringle 4 interaction is estimated to be Ka approximately 74 mM-1, which should be compared with Ka approximately 145 mM-1 previously measured for kringle 1 under identical conditions. It is proposed that residues in the proximity of the Cys80-Cys1 disulfide bridge are proximal to, or form part of, the lysine-binding site.     

Kringle 4 from human plasminogen: a proton magnetic resonance study via two-dimensional photochemically induced dynamic nuclear polarization spectroscopy

Two-dimensional (2D) proton magnetic resonance techniques used in conjunction with laser photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy have been applied to studying the kringle 4 domain from human plasminogen at 360 MHz. Out of 11 potential CIDNP-sensitive aromatic side chains, only 5 (His3, Tyr41, Tyr50, Trp72, and Tyr74) appear to be accessible to 3-(carboxymethyl)lumiflavin, the dye used to photogenerate spin polarization. Of these, Trp72 and Tyr74 are known to be at, or near, the lysine-binding site. The spin-spin scalar (J) and phase-sensitive dipolar (Overhauser) connectivities in the 2D experiments yield absolute assignments for the aromatic signals stemming from the exposed tyrosyl and tryptophanyl rings. Moreover, a number of side-chain H beta resonances can be identified and assigned to specific types of aromatic amino acid residues.     

Binding of the NG2 proteoglycan to kringle domains modulates the functional properties of angiostatin and plasmin(ogen)

Interactions of the developmentally regulated chondroitin sulfate proteoglycan NG2 with human plasminogen and kringle domain-containing plasminogen fragments have been analyzed by solid-phase immunoassays and by surface plasmon resonance. In immunoassays, the core protein of NG2 binds specifically and saturably to plasminogen, which consists of five kringle domains and a serine protease domain, and to angiostatin, which contains plasminogen kringle domains 1-3. Apparent dissociation constants for these interactions range from 12 to 75 nm. Additional evidence for NG2 interaction with kringle domains comes from its binding to plasminogen kringle domain 4 and to miniplasminogen (kringle domain 5 plus the protease domain) with apparent dissociation constants in the 18-71 nm range. Inhibition of plasminogen and angiostatin binding to NG2 by 6-aminohexanoic acid suggests that lysine binding sites are involved in kringle interaction with NG2. The interaction of NG2 with plasminogen and angiostatin has very interesting functional consequences. 1) Soluble NG2 significantly enhances the activation of plasminogen by urokinase type plasminogen activator. 2) The antagonistic effect of angiostatin on endothelial cell proliferation is inhibited by soluble NG2. Both of these effects of NG2 should make the proteoglycan a positive regulator of the cell migration and proliferation required for angiogenesis.