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.     

Solution structure of the kringle 4 domain from human plasminogen by 1H nuclear magnetic resonance spectroscopy and distance geometry

Kringle 4 is an autonomous structural and folding domain within the proenzyme plasminogen. Homologous domains are found throughout the blood clotting and fibrinolytic proteins. In this paper, we present the almost complete assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the kringle 4 domain of human plasminogen. A detailed structural analysis has been completed. The sequential pattern of nuclear Overhauser enhancements indicated little regular secondary structure but rather a series of turns and loops connecting beta-strands. A small stretch of antiparallel beta-sheet was identified between the residues 61 to 63 and 71 to 73 and the close proximity of other strands was determined from two-dimensional nuclear Overhauser enhancement spectra. Slowly exchanging amide (NH) resonances were found to be associated with residues of the beta-sheet and neighbouring strands that support the hydrophobic core of the domain. A total of 526 interproton distance constraints and two hydrogen bonds were specified as input to the distance geometry program DISGEO. Tertiary structures were produced that were consistent with the n.m.r. data. The structures were compared with that of our earlier model based on n.m.r. studies and with that of prothrombin fragment 1 determined crystallographically.     

Examination of the secondary structure of the kringle 4 domain of human plasminogen

The structure of a small region of human plasminogen (F4), consisting of amino acid residues Val354-Ala439 and containing its kringle 4 (K4) domain (residues Cys357-Cys434), has been predicted from Chou-Fasman calculations and hydropathy profiles, and compared to circular dichroism (CD) measurements on the isolated fragment. Calculations, by the Chou-Fasman method, of the probabilities of various types of secondary structures that exist in this region reveal that no helical structures are present. Of the total of 86 amino acid residues present in this K4-containing peptide region, 37% can adopt conformations of beta-pleated sheets, 48% of the amino acids can exist in beta-turns, and 15% of the residues can be present as coils. The structure of F4 in dilute aqueous solution has been experimentally evaluated by CD measurements. At pH = 7.4, in dilute salt solutions, a total of 64% beta-structures, 30% beta-turns, and 6% coiled structures is estimated to be present in this peptide region. Consideration of the marginal stability of many of the conformational regions of F4, as predicted by Chou-Fasman calculations, suggests that secondary structural flexibility is present in this fragment, which could result in ready adoption of new conformations. The hydropathy profile of F4 has been determined and suggests that this polypeptide is highly hydrophilic, especially in the regions of residues His387-Tyr396 and Cys406-Lys413. Thus, it appears as though a large portion of the surface of F4 can be exposed to solvent in its native conformation.     

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.     

Ligand-binding effects on the kringle 4 domain from human plasminogen: a study by laser photo-CIDNP 1H-NMR spectroscopy

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) one-dimensional and two-dimensional (2D) 1H-NMR techniques have been applied to the study of the kringle 4 domain of human plasminogen both ligand-free and complexed to the antifibrinolytic drugs epsilon-aminocaproic acid and p-benzylaminesulfonic acid (BASA). A number of aromatic side-chains (His3, Trp72, Tyr41, Tyr50 and Tyr74) appear to be exposed and accessible to 3-N-carboxymethyl-lumiflavin, the photopolarizing flavin dye, both in the presence and in the absence of ligands. A lesser exposure is observed for the Trp25 and Trp62 indole groups in the presence of BASA. The spin-spin (J-coupling) and dipolar (Overhauser) connectivities in the 2D experiments afford absolute assignment of aromatic resonances for the above residues, as well as of those stemming from the Trp72 ring in the presence of BASA. Moreover, a number of H beta resonances can be identified and sorted according to specific types of amino acid residues.     

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.     

Ligand-binding effects on the kringle 4 domain from human plasminogen: a study by laser photo-CIDNP1H-NMR spectroscopy

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) one-dimensional and two-dimensional (2D)¹H-NMR techniques have been applied to the study of the kringle 4 domain of human plasminogen both ligand-free and complexed to the antifibrinolytic drugs ɛ-aminocaproic acid and p-benzylaminesulfonic acid (BASA). A number of aromatic side-chains (His³, Trp⁷², Tyr⁴¹, Tyr⁵⁰ and Tyr⁷⁴) appear to be exposed and accessible to 3-N-car☐ymethyl-lumiflavin, the photopolarizing flavin dye, both in the presence and in the absence of ligands. A lesser exposure is observed for the Trp²⁵ and Trp⁶² indole groups in the presence of BASA. The spin-spin (J-coupling) and dipolar (Overhauser) connectivities in the 2D experiments afford absolute assignment of aromatic resonances for the above residues, as well as of those stemming from the Trp⁷² ring in the presence of BASA. Moreover, a number of H^β resonances can be identified and sorted according to specific types of amino acid residues.     

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)     

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

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, N- and C-terminal analyses, and high-resolution 1H-NMR spectroscopy at both 300 MHz 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 1H-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 (Ka approximately equal to 1.7 mM -1) 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.     

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.     

Analysis and identification of aromatic signals in the proton magnetic resonance spectrum of the kringle 4 fragment from human plasminogen

The aromatic 1H NMR spectrum of the kringle 4 domain from human plasminogen has been reexamined in order to identify signals stemming from individual residues. Acid-base titration, nuclear Overhauser effect experiments, and two-dimensional correlated spectroscopies have been implemented in order to analyze the spectrum both in the presence and in the absence of ligands. All six histidyl imidazole singlets have been recognized and paired according to their common side-chain origin. A similar identification has been achieved for the three sets of tryptophanyl resonances, and for Trp-I, the correspondence between indole singlet and multiplets is unambiguously established. The single phenylalanyl side chain and all tyrosyl phenol spin systems have been identified. Titration experiments indicate that one or two of the tryptophans are in the vicinity of carboxyl groups. It is shown that the spectrum for one tyrosyl ring, Tyr-V, undetectable at approximately 300 MHz, becomes visible at 600 MHz, reflecting slow motion on the NMR time scale and a constrained location within the kringle. A simulation of the complete kringle 4 aromatic spectrum is included.     

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.     

Expression of kringle 5 domain of human plasminogen in Pichia pastoris

The kringle 5 domain of plasminogen exhibits potent inhibitory effect on endothelial cell proliferation. It can also cause cell cycle arrest and apoptosis of endothelia cell specifically, and shows promise in antiangiogenic therapy. It has been prepared via both proteolysis of native plasminogen and recombinant DNA methodologies. When expressed in E. coli, recombinant, kringle 5 deposited mainly as inactive, insoluble inclusion bodies and the refolding yield was also low. In the present study, human kringle 5 encoding gene was cloned into secretory plasmid pPIC9K and then integrated into Pichia pastoris genome for expression. On methanol induction, biologically active recombinant kringle 5 was expressed and secreted into the culture medium by the integrated Pichia pastoris with the expression level around 30mg/L of yeast culture. After a simple and economical three-step purification protocol, namely precipitation, DEAE ion exchange chromatography, and gel filtration, the recombinant kringle 5 was purified to homogeneity, with the yield of 7.5 mg/liter yeast culture.     

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.     

Crystal structure of the kringle 2 domain of tissue plasminogen activator at 2.4-A resolution.

The crystal structure of the kringle 2 domain of tissue plasminogen activator was determined and refined at a resolution of 2.43 A. The overall fold of the molecule is similar to that of prothrombin kringle 1 and plasminogen kringle 4; however, there are differences in the lysine binding pocket, and two looping regions, which include insertions in kringle 2, take on very different conformations. Based on a comparison of the overall structural homology between kringle 2 and kringle 4, a new sequence alignment for kringle domains is proposed that results in a division of kringle domains into two groups, consistent with their proposed evolutionary relation. The crystal structure shows a strong interaction between a lysine residue of one molecule and the lysine/fibrin binding pocket of a noncrystallographically related neighbor. This interaction represents a good model of a bound protein ligand and is the first such ligand that has been observed in a kringle binding pocket. The structure shows an intricate network of interactions both among the binding pocket residues and between binding pocket residues and the lysine ligand. A lysine side chain is identified as the positively charged group positioned to interact with the carboxylate of lysine and lysine analogue ligands. In addition, a chloride ion is located in the kringle-kringle interface and contributes to the observed interaction between kringle molecules.     

The effects of ligand binding on the backbone dynamics of the kringle 1 domain of human plasminogen

The internal motions of the backbone nitrogen atoms of the kringle 1 domain of human plasminogen (K1(Pg)) were examined in the absence and presence of the ligand, epsilon-aminocaproic acid. These dynamic properties were determined from (15)N NMR relaxation data in terms of the extended model-free parameters. The model of isotropic reorientation was found sufficient to account for overall molecular tumbling for both apo and EACA-bound K1(Pg). The global rotational correlation time (tau(m)) for apo-K1(Pg) was 5.87(+/-0.01) ns, while the tau(m) for ligand-bound K1(Pg) was 5.20(+/-0.01) ns, suggesting that perhaps some small degree of aggregation occurred in the apo form of the kringle module. Complexation of K1(Pg) with ligand mainly reduced those internal motions that occurred on a 100 ps to 5 ns time-scale. The magnitude of the chemical exchange was also attenuated upon ligand binding. These data are consistent with studies employing other approaches that suggest that the binding pocket is preformed in K1(Pg).     

Ligand specificity of human plasminogen kringle 4.

The ligand specificity of the human plasminogen kringle 4 was characterized in terms of ligand size, aromatic/aliphatic character, and ionic charge distribution. The binding of the following ligands was investigated via 1H NMR spectroscopy, and their equilibrium association constants (Ka) were determined: (1) p-aminomethylbenzoic acid (Ka approximately 4.8 mM-1), (2) benzylamine (Ka approximately 0.2 mM-1), (3) l-aminohexane (Ka approximately 0.07 mM-1), (4) 7-aminoheptanoic acid (Ka approximately 6.6 mM-1), (5) 5-aminopentanoic acid (Ka approximately 16 mM-1), (6) N alpha-acetyl-L-arginine (Ka approximately 0.3 mM-1), and (7) N alpha-acetyl-L-arginine methyl ester (Ka approximately 0.08 mM-1). Benzamidine and L-arginine do not bind measurably to kringle 4. We have also established that 1-hexanoic acid and 4-methylbenzoic acid do not interact significantly with kringle 4 (Ka less than 0.05 mM-1). The Trp62 resonances were found to be quite sensitive to aromatic ligands as well as to aliphatic ligand length. Phe64 is similarly sensitive to the ligand aromatic/aliphatic character and chain length and to the identity of the ligand anionic group. His31 and His33 do not respond significantly to variations in ligand structure, although they are perturbed by aromatic and aliphatic effectors. The perturbations induced by the arginine derivatives on these residues show that these compounds interact with the lysine-binding site (LBS) of kringle 4. The LBS was further characterized using 2D NMR studies of a kringle 4/trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA) complex. A complete assignment of the AMCHA spectrum in the bound state was achieved. This enabled the unambiguous identification of intermolecular contact points between the central AMCHA protons and Trp62 and Trp72. A model based on the X-ray crystallographic structure of kringle 4, incorporating these constraints, has been derived.     

Proton nuclear magnetic resonance study of cobrotoxin.

Cobrotoxin (Mr 6949), which binds tightly to the acetylcholine receptors, contains no phenylalanines and only two histidines, two tyrosines, and one tryptophan that result in well-resolved aromatic proton resonances in D2O at 360 MHz. His-32, Tyr-25, and the Trp are essential for toxicity and may interact with the acetylcholine receptor. We assign two titratable resonances (pKa = 5.1) at delta = 9.0 and 7.5 ppm at pH 2.5 and at 7.7 and 7.1 ppm at pH 9.5 to the C-2 and C-4 ring protons, respectively, of His-4. Two other titratable resonances (pKa = 5.7) at delta = 8.8 and 6.9 ppm at pH 2.5 and at 7.8 and 6.7 ppm at pH 9.5 are assigned to the C-2 and C-4 ring protons of His-32, respectively. The differences in delta values of the two histidines reflect chemically different microenvironments while their low pKa values could arise from nearby positive charges. A methyl resonance gradually shifts upfield to delta approximately 0.4 ppm as His-4 is deprotonated and is tentatively assigned to the methyl group of Thr-14 or Thr-15 which, from published X-ray studies of neurotoxins, are located in the vicinity of His-4. Further, we have identified the aromatic resonances of the invariant tryptophan and individual tyrosines and the methyl resonance of one of the two isoleucines in the molecule. Several broad nontitrating resonances of labile protons which disappear at pH greater than 9 may arise from amide groups of the beta sheet in cobrotoxin.