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

Demarco, A; Petros, Am; Llinás, Miguel; Kaptein, Robert; Boelens, Rolf

doi:10.1016/0167-4838(89)90151-9

Cited by 19 publications

(9 citation statements)

References 30 publications

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“…For the Trp indole spin systems of the PGN kringles, the most intense cross-peaks are displayed by Trp72, consistent with a rather mobile side chain. Chemical modification (Hochschwender & Laursen, 1981) and photo-CIDNP (De Marco et al, 1989) experiments on PGN kringle 4 indicate that the Trp72 side chain is accessible to solvent. Also, the COSY connectivity pattern of both the Trp-III spin system in t-PA kringle 2 and the Trp72 spin system in the PGN kringle 4 variants is similar (Figure 7).…”

Section: Resultsmentioning

confidence: 98%

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: Resultsmentioning

confidence: 98%

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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“…3A), cross-polarized by the flavin which we have shown to specifically interact with Phe 31 and Trp 23. Several examples are reported of indirect polarization of residues in close contact with strongly polarized aromatic residues [22][23][24]. In particular, photo-CIDNP effects were observed in the presence of specific binding interactions between flavin and the protein [24,25].…”

Section: Resultsmentioning

confidence: 99%

“…Several examples are reported of indirect polarization of residues in close contact with strongly polarized aromatic residues [22][23][24]. In particular, photo-CIDNP effects were observed in the presence of specific binding interactions between flavin and the protein [24,25]. In these examples the binding constant was not strong enough to prevent the radicals in the radical pair to separate ( [10] and references therein) and photo-CIDNP effects could be observed.…”

Section: Resultsmentioning

confidence: 99%

¹H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp²³ and Phe³¹ in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus

Consonni¹,

Limiroli

Molinari

et al. 1995

FEBS Letters

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“…The [KlPg] (Menhart et al, 1991;Hoover et al, 1993) and [K4Pg] (Menhart et al, 1993) domains, as well as a number of mutants of these modules, have been expressed, and some general features of their co-amino acid binding sites have been identified. These advances, coupled with the determination of X-ray crystal structures of [K1 Pg] (Wu et al, 1994) and of the EACA/[K4Pg] complex (Wu et al, 1991), along with high-resolution multidimensional NMR investigations of these domains (De Marco et al, 1986Marco et al, , 1989Thewes et al, 1987;Rejante & Llinas, 1994), clearly implicate certain amino acid residues common to all co-amino acid binding kringles in stabilizing ligand/kringle binding. One amino acid residue critical to binding of EACA in all kringles is an Asp residue at sequence positions homologous to Asp56 (numbering from the first Cys residue of the kringle) of [KlPg] (Asp139 in Pg), and this was the focus of the mutations designed herein.…”

mentioning

confidence: 99%

Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation

Menhart

Hoover²,

McCance³

1995

Biochemistry

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In order to identify the individual contributions of the kringle (K) domains of human plasminogen (Pg) to the epsilon-aminocaproic acid (EACA) induced stimulation of Pg activation by low-molecular-weight urokinase-type plasminogen activator (LMW-uPA) and inhibition of this same activation by Cl-, we constructed the most conservative recombinant- (r-) Pg mutants possible that would greatly reduce the strength of the EACA binding site in the omega-amino acid binding kringles, [K1Pg] ([D139-->N]r-Pg), [K4Pg] ([D413-->N]r-Pg), and [K5Pg] ([D515--N]r-Pg). In each case, this involved mutation of a critical Asp (to Asn) within these three kringle domains in intact Pg. The three r-mutants were expressed in r-baculovirus-infected lepidopteran insect (Trichoplusia ni) cells. In the presence of Cl-, the positive activation effector, EACA, first stimulated and then inhibited the LMW-uPA-catalyzed initial activation of wild-type (wt) r-[Glu1]Pg and, to a lesser extent, the [K5Pg] mutant, [D518-->N/Glu1]r-Pg. The concentration of EACA that produced 50% stimulation of activation (C50) occurred at 3.3 mM for wtr-[Glu1]Pg and at 0.7 mM for [D518-->N/Glu1]r-Pg. Subsequent inhibition by EACA occurred with a C50 of approximately 15 mM and is likely due to inhibition of the amidolytic activity of plasmin generated during the activation. Similar initial activation rates of both [D139-->N]r-Pg and [D413N]r-Pg did not display this initial EACA-mediated stimulatory phase but did undergo ultimate inhibition with a C50 for this process that was similar to wtr-[Glu1]Pg and [D518-->N/Glu1]r-Pg.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Cited by 19 publications

References 30 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

¹H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp²³ and Phe³¹ in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus

Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation

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

Cited by 19 publications

References 30 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

1H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp23 and Phe31 in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus

Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation

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¹H‐NMR and photo‐CIDNP spectroscopies show a possible role for Trp²³ and Phe³¹ in nucleic acid binding by P2 ribonuclease from the archaeon Sulfolobus solfataricus