Proton magnetic resonance study of kringle 1 from human plasminogen. Insights into the domain structure

The aromatic H NMR spectrum of the kringle 1 domain from human plasminogen has been investigated by proton Overhauser experiments, acid-base titration, and two-dimensional chemical shift correlated spectroscopy. Spin-echo and pH response experiments lead to the identification of the N-terminal Tyr-3 phenol ring signals. The connectivities among the tryptophanyl aromatic protons have been established and sets of singlet-doublet-triplet resonances stemming from each of the two indole groups sorted according to their common side chain origin. Similarly, the four histidyl singlets have been identified and paired per imidazole group. From their pH responses, it is indicated that a histidyl (His31) and a tryptophanyl (Trp-II) residue are placed in the neighborhood of carboxyl groups. The high-field chemical shifts observed for proton resonances of the ligand epsilon-aminocaproic acid upon binding to kringle 1 indicate that the ligand-binding site is rich in aromatic components. Overhauser experiments reveal that Leu46 is surrounded by a cluster of interacting aromatic side chains, which includes Trp25, Phe36, His41, Trp62, and Tyr64, and define a hydrophobic region contiguous to the kringle lysine-binding site. Relative internuclear distances have been estimated for aromatic H-atoms in the vicinity of Leu46 by reference to one of the latter's CH3 sigma, sigma' groups. Some of the connectives have previously been found for Leu46 in kringle 4 which further supports the idea of a common structure for the homologous domains.     

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 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.     

The refined structure of the epsilon-aminocaproic acid complex of human plasminogen kringle 4.

The crystallographic structure of the plasminogen kringle 4-epsilon-aminocaproic acid (ACA) complex (K4-ACA) has been solved by molecular replacement rotation-translation methods utilizing the refined apo-K4 structure as a search model (Mulichak et al., 1991), and it has been refined to an R value of 0.148 at 2.25-A resolution. The K4-ACA structure consists of two interkringle residues, the kringle along with the ACA ligand, and 106 water molecules. The lysine-binding site has been confirmed to be a relatively open and shallow depression, lined by aromatic rings of Trp62, Phe64, and Trp72, which provide a highly nonpolar environment between doubly charged anionic and cationic centers formed by Asp55/Asp57 and Lys35/Arg71. A zwitterionic ACA ligand molecule is held by hydrogen-bonded ion pair interactions and van der Waals contacts between the charged centers. The lysine-binding site of apo-K4 and K4-ACA have been compared: the rms differences in main-chain and side-chain positions are 0.25 and 0.69 A, respectively, both practically within error of the determinations. The largest deviations in the binding site are due to different crystal packing interactions. Thus, the lysine-binding site appears to be preformed, and lysine binding does not require conformational changes of the host. The results of NMR studies of lysine binding with K4 are correlated with the structure of K4-ACA and agree well.     

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.     

Analysis of ligand-binding to the kringle 4 fragment from human plasminogen

The interaction of the isolated human plasminogen kringle 4 with the four -amino acid ligands -aminocaproic acid (ACA), N-acetyl-l-lysine (AcLys), trans-aminomethyl(cyclohexane)carboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) has been further characterized by ¹H-NMR spectroscopy at 300 and 600 MHz. Pronounced high-field shifts, reaching 3 ppm, are observed for AMCHA resonances upon binding to kringle 4, which underscores the relevance of ligand lipophilic interactions with aromatic side chains at the binding site. Ligand titration curves for the nine His and Trp singlets found in the kringle 4 aromatic spectrum reveal a striking uniformity in the kringle response to the various ligands. The average binding curves exhibit a clear Langmuir absorption isotherm saturation profile and the data were analyzed under the assumption of one (high affinity) binding site per kringle. Equilibrium association constants (K _a ) and first order dissociation rate constants (k _off) were derived from linearized expressions of the Langmuir isotherm and of the spectral line-shapes, respectively. The results for the four ligands, at 295 K, pH^* 7.2, indicate that: (a) AMCHA exhibits the strongest binding (K _a =159 mM ^-1) and ACA the weakest (K _a =21 mM ^–1) with AcLys and BASA falling in between; (b) ACA dissociates readily (k _off = 5.3 × 10³ s^–1) and AMCHA associates the fastest (k _off = 2.0 × 10⁸ M ^–1 s^–1) while the kinetics for BASA exchange is relatively slow (k _off = 0.8 × 10³ s^–1, k _on = 0.6 × 10⁸ M ^–1s^–1); (c) the ligand-binding kinetics is close to diffussion-controlled.Abbreviations ACA -aminocaproic acid - AcLys N-acetyl-l-lysine - AMCHA t-aminomethyl(cyclohexane)carboxylic acid - BASA p-benzylaminesulfonic acid - K4 kringle 4 - NOE nuclear Overhauser effect - ppm parts-per-million - pH^* glass electrode pH reading uncorrected for deuterium isotope effects - K _a ligand-kringle 4 equilibrium association constant - k _off ligand-kringle 4 dissociation rate constant - k _on ligand-kringle 4 association rate constant     

Characterization of the low-field proton magnetic resonance spectrum of plasminogen kringle 4 via selective Overhauser experiments in 1H2O

The low-field 1H NMR spectrum of the kringle 4 domain of human plasminogen has been investigated at 300 and 600 MHz for the protein dissolved in 1H2O. The spectrum exhibits six well-resolved resonances, spanning the 9.8 approximately less than delta approximately less than 13 ppm chemical shift range, which arise from exchange-labile H atoms. The acid-base response of the six resonances was monitored in order to characterize the signals in terms of their pH titration profiles. The sensitivity of the low-field resonances to kringle binding the antifibrinolytic ligands N alpha-acetyl-L-lysine and p-benzylaminesulfonic acid was also investigated. The lowest field resonance, at 12.6 ppm, is a doublet of J approximately 7.9 Hz, a splitting that is unprecedented for His or Trp ring NH signals. Selective Overhauser experiments centered on the exchangeable proton transitions identify four of the other resonances as stemming from the His31, His33, Trp I, and Trp II side-chain NH groups, where the latter two are, as yet, not definitely assigned to the specific residues, Trp25 and Trp62. The relative narrowness of the His imidazole NH signals indicates that the two rings are sterically shielded from direct water accessibility. In particular, the His33 NH site appears to be the most protected. The Overhauser evidence conclusively shows that the two identified exchangeable His ring proton signals arise from imidazole NH3 sites rather than from the NH1 tautomers. Similarly, these experiments lead to an unambigous characterization of the corresponding Trp aromatic CH spin systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal and molecular structure of human plasminogen kringle 4 refined at 1.9-A resolution.

The crystal structure of human plasminogen kringle 4 (PGK4) has been solved by molecular replacement using the bovine prothrombin kringle 1 (PTK1) structure as a model and refined by restrained least-squares methods to an R factor of 14.2% at 1.9-A resolution. The K4 structure is similar to that of PTK1, and an insertion of one residue at position 59 of the latter has minimal effect on the protein folding. The PGK4 structure is highly stabilized by an internal hydrophobic core and an extensive hydrogen-bonding network. Features new to this kringle include a cis peptide bond at Pro30 and the presence of two alternate, perpendicular, and equally occupied orientations for the Cys75 side chain. The K4 lysine-binding site consists of a hydrophobic trough formed by the Trp62 and Trp72 indole rings, with anionic (Asp55/Asp57) and cationic (Lys35/Arg71) charge pairs at either end. With the adjacent Asp5 and Arg32 residues, these result in triply charged anionic and cationic clusters (pH of crystals at 6.0), which, in addition to the unusually high accessibility of the Trp72 side chain, serve as an obvious marker of the binding site on the K4 surface. A complex intermolecular interaction occurs between the binding sites of symmetry-related molecules involving a highly ordered sulfate anion of solvation in which the Arg32 side chain of a neighboring kringle occupies the binding site.     

Two-dimensional 1H-NMR studies of the solution structure of plasminogen kringle 4

Native kringle 4 from human plasminogen has been studied by two-dimensional 1H-NMR methods in order to obtain new structural information about the kringle fold. Two-dimensional scalar correlated spectroscopy (COSY), two-dimensional dipolar correlated spectroscopy (NOESY) and two-dimensional relayed coherance transfer spectroscopy (RCT) experiments were recorded, allowing most resonances arising from the aromatic and methyl-containing residues to be assigned in the spectrum. From an analysis of NOE data, a small segment of double-stranded beta-sheet has been identified near residues Phe63 and Thr64. Further analysis of the NOESY spectrum has allowed detailed study of the conformation of sidechains located in regions near Leu45 and Val69. A model has been constructed of the polypeptide segment comprising residues 40-49 which accounts for the observed NOE interactions.     

Secondary structure predictions of human plasminogen and the bovine prothrombin kringle loops

Secondary structural predictions, based upon the statistical methodology of Chou and Fasman, for the kringle loops of human plasminogen and bovine prothrombin suggest a "winding staircase" pattern of beta-turns, spaced by short regions of ordered and coil structures. Analysis of the predicted structures of the regions containing the two His (113 and 387) and Asp (136 and 410) residues in plasminogen kringles 1 and 4, which have been found to be important in binding the ligand, epsilon-aminocaproic acid, shows that all are localized at the same positions on beta-turns. In addition, both of the two Asp residues occur at the end of homologous nonapeptide regions common to all of the five human plasminogen and two bovine prothrombin kringles, indicating evolutionary conservation to preserve biologically critical conformations. Examination of the protein conformation in the region of Asn288, the residue which is glycosylated in one of the two circulating variants of human plasminogen, shows that it most likely exists in a position which may present topographical hindrance to post-translational attachment of carbohydrate, thus, possibly, explaining the incomplete glycosylation of human plasminogen with complex-type carbohydrate.     

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.     

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.     

Macro- and micro-stabilities of the kringle 4 domain from plasminogen. The effect of ligand binding.

1H-NMR spectra of kringle 4 from human plasminogen have been recorded over wide pH* and temperature ranges, both in the presence and in the absence of p-benzylaminesulfonic acid (BASA). Several resonances exhibit chemical shift differences between kringle folded and unfolded forms which are sufficiently well resolved to allow for a determination of equilibrium Van't Hoff enthalpies and entropies for unfolding. The interaction with BASA shifts the kringle unfolding temperature from approximately 335 degrees K to approximately 343 degrees K. The pH* range of stability is also wider for the complex than for the free kringle: in the acidic range the pH* of half-unfolding, pHu*, is decreased from 2.8 for the unligated polypeptide to approximately 2.0 in the presence of BASA, while in the basic range pHu*, shifts from approximately 10.8 to 11.5. However, in contrast with what is observed at acidic pH*, unfolding at basic pH* leads to irreversible denaturation and exhibits a sharp, order-disorder transition both in the presence and in the absence of ligand. The structural stabilization conferred by the ligand is accompanied by a drastic reduction of the average rate of 1H-2H exchange in 2H2O under conditions that preclude a major cooperative unfolding. Thus, macro- and micro-stabilities of kringle domains appear to be highly correlated.     

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 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)     

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.     

The lysine binding sites of human plasminogen. Evidence for a critical tryptophan in the binding site of kringle 4

Chemical modification of human degraded form of plasminogen with NH2-terminal lysine (Lys-plasminogen) and the elastase fragments kringle 1 + 2 + 3 and kringle 4 with the tryptophan reagent [14C]dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide results in the incorporation of label and the parallel loss of lysine binding ability. In the case of kringle 4, only one-half of the lysine binding sites could be inactivated, but the modified and unmodified forms could be separated by affinity chromatography. The modified form contained 1 mol of 2-hydroxy-5-nitrobenzyl groups/mol of kringle 4 and did not bind to lysine-Sepharose. Lysine analogs such as 6-aminohexanoic acid protected kringle 4 against modification. Peptide-mapping studies on this form showed that essentially all of the label was in two chymotryptic peptides containing a tryptophan corresponding to Trp426 in the plasminogen sequence. Competition experiments with anti-kringle 4 antibodies having an affinity for the lysine binding site showed that the binding of 2-hydroxy-5-nitrobenzyl-kringle 4 to antibodies was about 10 times weaker than for unmodified kringle 4. These results indicate that the integrity of specific tryptophan residue is critical to the binding of lysine and related amino acids to kringle 4of human plasminogen.     

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.     

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.     

Proton nuclear Overhauser effect study of the heme active site structure of chloroperoxidase.

Chloroperoxidase, a glycoprotein from the mold Caldariomyces fumago, has been investigated in its ferric low-spin cyanide-ligated form through use of nuclear Overhauser effect (NOE) spectroscopy to provide information on the heme pocket electronic/molecular structure. Spin-lattice relaxation times for the hyperfine-shifted heme resonances were found to be three times less than those in horseradish peroxidase. This must reflect a slower electronic relaxation rate for chloroperoxidase than for horseradish peroxidase as a consequence of axial ligation of cysteine in the former versus histidine in the latter enzyme. Isoenzymes A1 and A2 of chloroperoxidase show the largest chemical shift differences near the heme propionate on the basis of NOE measurements. This suggests that the primary structure differences for the two isoenzymes are communicated to the heme group through the ring propionate substituents. A downfield peak has been detected in chloroperoxidase with chemical shift, T1, and line width characteristics similar to those of the C epsilon-H proton of the distal histidine residue. The NOE pattern and T1's of the peaks in the 0.0 to -5.0 ppm upfield region are consistent with the presence of an arginine amino acid residue in the heme pocket near either the 1-CH3 or 3-CH3 group. Existence of catalytically important distal histidine and arginine amino acid residues in chloroperoxidase shows it to be structurally similar to peroxidases rather than to the often compared monooxygenase, cytochrome P-450. This result supports the earlier conclusions of Sono et al. [Sono, M., Dawson, J.H., Hall, K., & Hager, L.P. (1986) Biochemistry 25, 347-356].