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

Petros, Am; Gyenes, Marianne; Patthy, László; Llinás, Miguel

doi:10.1016/0003-9861(88)90585-1

Cited by 12 publications

(7 citation statements)

References 27 publications

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“…A Tyr spectrum containing four one-proton doublets reveals a slow flip dynamics for the corresponding phenol ring. A similar pattern has been observed for Tyr9 in PGN kringle 4 homologues (De Marco et al, 1985b;Ramesh et al, 1986;Petros et al, 1988b). Therefore, we assign Tyr-V in t-PA kringle 2 to Tyr9.…”

Section: Resultssupporting

confidence: 82%

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

Byeon

Kelley²,

Llinás³

1989

Biochemistry

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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)

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Section: Resultssupporting

confidence: 82%

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

Byeon

Kelley²,

Llinás³

1989

Biochemistry

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“…In the presence of the other ligands, and for ligand-free K4, the Tyr9 resonances appear to be too broad to be detectable. It has already been shown in our laboratory (De Marco et al, 1985b;Ramesh et al, 1986;Petros et al, 1988b) that the appearance of Tyr9 signals is highly dependent on temperature, observational frequency, and ligand presence. At 600 MHz, 293 K, with no added ligand, the Tyr9 phenol ring protons exhibit an ABCD-type spectral pattern.…”

Section: S2(ppm)mentioning

confidence: 91%

Proton NMR studies of aliphatic ligand binding to human plasminogen kringle 4

Petros¹,

Ramesh²,

Llinás³

1989

Biochemistry

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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)

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“…On the other hand, although a role in ligandbinding has been proposed for His31 (35), this side chain appears to be too far removed from the site to participate directly. Ligandbinding has also been reported to involve the flip rate of Tyr9 (8,36,37); however, there is no obvious reason for this observation based on the crystal structure, in which Tyr9 appears to only communicate with the binding site very, very remotely (23). The amino terminal of the K4 peptide chain, which can partially restrict the Tyr9 side chain conformation and which is not fully determined in the crystallographic structure, may be somehow involved in this effect.…”

Section: Kringle 4 Complexed With Ac Amentioning

confidence: 99%

The Structures of Domains of Blood Proteins

Tulinsky¹

1991

Thromb Haemost

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

Cited by 12 publications

References 27 publications

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

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

Proton NMR studies of aliphatic ligand binding to human plasminogen kringle 4

The Structures of Domains of Blood Proteins

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