6-Aminohexanoic Acid

14,000,000 Leading Edge Experts on the ideXlab platform

Scan Science and Technology

Contact Leading Edge Experts & Companies

Scan Science and Technology

Contact Leading Edge Experts & Companies

The Experts below are selected from a list of 174 Experts worldwide ranked by ideXlab platform

Francis J. Castellino - One of the best experts on this subject based on the ideXlab platform.

  • enhancement through mutagenesis of the binding of the isolated kringle 2 domain of human plasminogen to ω amino Acid ligands and to an internal sequence of a streptococcal surface protein
    Journal of Biological Chemistry, 1999
    Co-Authors: Stephanie L Nilsen, Mary Prorok, Francis J. Castellino
    Abstract:

    Abstract In the background of the recombinant K2 module of human plasminogen (K2Pg), a triple mutant, K2Pg[C4G/E56D/L72Y], was generated and expressed inPichia pastoris cells in yields exceeding 100 mg/liter. The binding affinities of a series of lysine analogs, viz.4-aminobutyric Acid, 5-aminopentanoic Acid, e-aminocaproic Acid, 7-aminoheptanoic Acid, andt-4-aminomethylcyclohexane-1-carboxylic Acid, to this mutant were measured and showed up to a 15-fold tighter interaction, as compared with wild-type K2Pg (K2Pg[C4G]). The variant, K2Pg[C4G/E56D], afforded up to a 4-fold increase in the binding affinity to these same ligands, whereas the K2Pg[C4G/L72Y] mutant decreased the same affinities up to 5-fold, as compared with K2Pg[C4G]. The thermal stability of K2Pg[C4G/E56D/L72Y] was increased by approximately 13 °C, as compared with K2Pg[C4G]. The functional consequence of up-regulating the lysine binding property of K2Pg was explored, as reflected by its ability to interact with an internal sequence of a plasminogen-binding protein (PAM) on the surface of group A streptococci. A 30-mer peptide of PAM, containing its K2Pg-specific binding region, was synthesized, and its binding to each mutant of K2Pg was assessed. Only a slight enhancement in peptide binding was observed for K2Pg[C4G/E56D], compared with K2Pg[C4G] (K d = 460 nm). A 5-fold decrease in binding affinity was observed for K2Pg[C4G/L72Y] (K d = 2200 nm). However, a 12-fold enhancement in binding to this peptide was observed for K2Pg[C4G/E56D/L72Y] (K d = 37 nm). Results of these PAM peptide binding studies parallel results of ω-amino Acid binding to these K2Pg mutants, indicating that the high affinity PAM binding by plasminogen, mediated exclusively through K2Pg, occurs through its lysine-binding site. This conclusion is supported by the 100-fold decrease in PAM peptide binding to K2Pg[C4G/E56D/L72Y] in the presence of 50 mm 6-Aminohexanoic Acid. Finally, a thermodynamic analysis of PAM peptide binding to each of these mutants reveals that the positions Asp56 and Tyr72 in the K2Pg[C4G/E56D/L72Y] mutant are synergistically coupled in terms of their contribution to the enhancement of PAM peptide binding.

  • involvement of tyrosine 76 of the kringle 2 domain of tissue type plasminogen activator in its thermal stability and its omega amino Acid ligand binding site
    Biochemistry, 1994
    Co-Authors: V S De Serrano, Francis J. Castellino
    Abstract:

    : A series of conservative and radical mutations have been made at an aromatic residue, Y76, of the isolated kringle 2 domain of tissue-type plasminogen activator ([K2tPA]) in order to assess the importance of this residue in the ligand binding properties and structural stability of this protein domain. We have successfully expressed in Escherichia coli r-[K2tPA] variants with the following amino Acid mutations at Y76: Y76-->A, Y76-->E, Y76-->F, Y76-->K, Y76-->L, Y76-->Q, and Y76-->W. The binding constants of 6-Aminohexanoic Acid (EACA) and 7-aminoheptanoic Acid (7-AHpA) to each of these mutants were investigated by titration of the alterations in intrinsic fluorescence of the mutant kringles with these amino Acid ligands. Compared to the wild-type kringle (r-[K2tPA]), which possessed dissociation constants (Kd) of 43 and 6 microM, respectively, for EACA and 7-AHpA, only the Y76-->E mutant displayed a substantially increased Kd value for these amino Acids, viz., 117 microM for 7-AHpA. More moderate increases in this parameter were observed for the Y76-->A and Y76-->K variants (2-3-fold increases in the Kd), with no significant differences noted in the cases of Y76-->L, Y76-->Q, and Y76-->W. A most interesting observation was made with the Y76-->F mutant, which showed a 4-6-fold reduction in the Kd for these amino Acid ligands. The conformations of all of the mutants were less stable than that of wtr-[K2tPA], as revealed by thermal denaturation studies, suggesting that a Y at sequence position 76 is of importance to the conformational stability of this kringle domain.(ABSTRACT TRUNCATED AT 250 WORDS)

  • role of the strictly conserved tryptophan 25 residue in the stabilization of the structure and in the ligand binding properties of the kringle 2 domain of tissue type plasminogen activator
    Biochemistry, 1994
    Co-Authors: V S De Serrano, Francis J. Castellino
    Abstract:

    : The involvement of the strictly conserved tryptophan-25 (W25) residue in the structural stability and omega-amino Acid ligand binding properties of the recombinant (r) kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) has been investigated. Two conservative mutants were constructed and expressed that contained W25-->F and W25-->Y substitutions. The binding (dissociation) constants (Kd) for three ligands, viz., 6-Aminohexanoic Acid (EACA), 7-aminoheptanoic Acid (7-AHpA), and L-lysine (Lys), to these polypeptides were determined by intrinsic fluorescence titrations. In the case of r-[K2tPA/W25F], the Kd values for these ligands were found to be 37, 16, and 89 microM for EACA, 7-AHpA, and Lys, respectively. For r-[K2tPA/W25Y], the Kd values for these same ligands were 64, 9, and 115 microM, respectively. The wild-type (wt) kringle domain possessed Kd values of 43, 6, and 85 microM for EACA, 7-AHpA, and Lys, respectively. The effect of these mutations on the stability of the r-[K2tPA] domain has been examined by differential scanning colorimetry. The temperature of maximum heat capacity (Tm) of wt-r-[K2tPA] (75.6 degrees C) was dramatically reduced to 50.8 and 58.0 degrees C for r-[K2tPA/W25F] and r-[K2tPA/W25Y], respectively. In the presence of EACA, the Tm values were increased to 86.1, 61.7, and 68.7 degrees C, respectively, indicating that EACA does interact with the r-[K2tPA] mutants and stabilizes their native conformations, similar to the case with wt-r-[K2tPA].(ABSTRACT TRUNCATED AT 250 WORDS)

  • amino Acids of the recombinant kringle 1 domain of human plasminogen that stabilize its interaction with omega amino Acids
    Biochemistry, 1993
    Co-Authors: Gordon J Hoover, Nick Menhart, Andrew Martin, Scott E Warder, Francis J. Castellino
    Abstract:

    : A series of strategically designed recombinant (r) mutants of the kringle 1 region of human plasminogen ([K1HPg]) have been constructed and the resulting gene products employed to reveal the identities of the residues that contribute to stabilization of the binding of omega-amino Acid ligands to this domain. On the basis of determinations of the binding constants of the ligands, 6-Aminohexanoic Acid and trans-4-(aminomethyl)cyclohexane-1-carboxylic Acid, to a variety of these mutants, we find that the anionic site of the polypeptide responsible for stabilization of the amino group of the ligands consists of both D54 and D56 and the cationic site of the polypeptide that interacts with the carboxylate group of the ligand is composed solely of R70. The main hydrophobic interactions that stabilize binding of these ligands, likely by interactions with the ligand hydrophobic regions, are principally due to W61, Y63, and Y71. The results obtained are consistent with conclusions that could be made from analysis of the X-ray crystal structure of r-[K1HPg] and from previous studies from this laboratory regarding the binding of ligands of this type to the kringle 2 region of tissue-type plasminogen activator ([K2tPA]). It thus appears as though a common ligand binding site has evolved in different kringles with ligand specificity differences between r-[K2tPA] and r-[K1HPg] perhaps explainable by the different nature of the cationic sites on these polypeptides that are involved in coordination to the ligand carboxylate groups.

  • specific anionic residues of the recombinant kringle 2 domain of tissue type plasminogen activator that are responsible for stabilization of its interaction with omega amino Acid ligands
    Biochemistry, 1993
    Co-Authors: V S De Serrano, Francis J. Castellino
    Abstract:

    : The involvement of specific aspartic Acid (D) and glutamic Acid (E) residues of the recombinant (r) kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) in stabilizing its interaction with omega-amino Acid ligands has been assessed by examination of these binding events subsequent to site-directed mutagenesis of the relevant amino Acid residues. We have expressed and purified nonconservative alanine (A) replacement mutants at the following amino Acid sequence locations in r-K2tPA:E17 (r-[K2tPA/E17A]), E75 (r-[K2tPA/E75A]), and D78 (r-[K2tPA/D78A]). More conservative E for D replacements were generated at the only other anionic (at neutral pH) amino Acids of r-[K2tPA], viz., D57 (r-[K2tPA/D57E]) and D59 (r-[K2tPA/D59E]). Each of these variant polypeptides was then utilized for binding investigations with a series of omega-amino Acids. No substantial differences were found in the binding constants (pH 8.0, 25 degrees C) for the ligands, 6-Aminohexanoic Acid (6-AHxA), 7-aminoheptanoic Acid (7-AHpA), L-lysine, and trans-(aminomethyl)cyclohexane-1-carboxylic Acid (AMCHA), among wild-type (wt) r-K2tPA, r-[K2tPA/E17A], r-[K2tPA/E75A], and r-[K2tPA/D78A]. On the other hand, dramatic effects on this same binding were observed in recombinant mutants with alterations at D57 and D59. In these cases, even with the most conservative replacements, i.e., r-[K2tPA/D57E] and r-[K2tPA/D59E], the Kd values for these ligands were increased approximately 3-6-fold and 18-85-fold, respectively. NMR analysis of these variants suggested that no substantial gross conformational changes occurred as a result of the mutations made, but some localized alterations in amino Acid microenvironments did take place.(ABSTRACT TRUNCATED AT 250 WORDS)

V S De Serrano - One of the best experts on this subject based on the ideXlab platform.

  • involvement of tyrosine 76 of the kringle 2 domain of tissue type plasminogen activator in its thermal stability and its omega amino Acid ligand binding site
    Biochemistry, 1994
    Co-Authors: V S De Serrano, Francis J. Castellino
    Abstract:

    : A series of conservative and radical mutations have been made at an aromatic residue, Y76, of the isolated kringle 2 domain of tissue-type plasminogen activator ([K2tPA]) in order to assess the importance of this residue in the ligand binding properties and structural stability of this protein domain. We have successfully expressed in Escherichia coli r-[K2tPA] variants with the following amino Acid mutations at Y76: Y76-->A, Y76-->E, Y76-->F, Y76-->K, Y76-->L, Y76-->Q, and Y76-->W. The binding constants of 6-Aminohexanoic Acid (EACA) and 7-aminoheptanoic Acid (7-AHpA) to each of these mutants were investigated by titration of the alterations in intrinsic fluorescence of the mutant kringles with these amino Acid ligands. Compared to the wild-type kringle (r-[K2tPA]), which possessed dissociation constants (Kd) of 43 and 6 microM, respectively, for EACA and 7-AHpA, only the Y76-->E mutant displayed a substantially increased Kd value for these amino Acids, viz., 117 microM for 7-AHpA. More moderate increases in this parameter were observed for the Y76-->A and Y76-->K variants (2-3-fold increases in the Kd), with no significant differences noted in the cases of Y76-->L, Y76-->Q, and Y76-->W. A most interesting observation was made with the Y76-->F mutant, which showed a 4-6-fold reduction in the Kd for these amino Acid ligands. The conformations of all of the mutants were less stable than that of wtr-[K2tPA], as revealed by thermal denaturation studies, suggesting that a Y at sequence position 76 is of importance to the conformational stability of this kringle domain.(ABSTRACT TRUNCATED AT 250 WORDS)

  • role of the strictly conserved tryptophan 25 residue in the stabilization of the structure and in the ligand binding properties of the kringle 2 domain of tissue type plasminogen activator
    Biochemistry, 1994
    Co-Authors: V S De Serrano, Francis J. Castellino
    Abstract:

    : The involvement of the strictly conserved tryptophan-25 (W25) residue in the structural stability and omega-amino Acid ligand binding properties of the recombinant (r) kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) has been investigated. Two conservative mutants were constructed and expressed that contained W25-->F and W25-->Y substitutions. The binding (dissociation) constants (Kd) for three ligands, viz., 6-Aminohexanoic Acid (EACA), 7-aminoheptanoic Acid (7-AHpA), and L-lysine (Lys), to these polypeptides were determined by intrinsic fluorescence titrations. In the case of r-[K2tPA/W25F], the Kd values for these ligands were found to be 37, 16, and 89 microM for EACA, 7-AHpA, and Lys, respectively. For r-[K2tPA/W25Y], the Kd values for these same ligands were 64, 9, and 115 microM, respectively. The wild-type (wt) kringle domain possessed Kd values of 43, 6, and 85 microM for EACA, 7-AHpA, and Lys, respectively. The effect of these mutations on the stability of the r-[K2tPA] domain has been examined by differential scanning colorimetry. The temperature of maximum heat capacity (Tm) of wt-r-[K2tPA] (75.6 degrees C) was dramatically reduced to 50.8 and 58.0 degrees C for r-[K2tPA/W25F] and r-[K2tPA/W25Y], respectively. In the presence of EACA, the Tm values were increased to 86.1, 61.7, and 68.7 degrees C, respectively, indicating that EACA does interact with the r-[K2tPA] mutants and stabilizes their native conformations, similar to the case with wt-r-[K2tPA].(ABSTRACT TRUNCATED AT 250 WORDS)

  • specific anionic residues of the recombinant kringle 2 domain of tissue type plasminogen activator that are responsible for stabilization of its interaction with omega amino Acid ligands
    Biochemistry, 1993
    Co-Authors: V S De Serrano, Francis J. Castellino
    Abstract:

    : The involvement of specific aspartic Acid (D) and glutamic Acid (E) residues of the recombinant (r) kringle 2 (K2) domain of tissue-type plasminogen activator (tPA) in stabilizing its interaction with omega-amino Acid ligands has been assessed by examination of these binding events subsequent to site-directed mutagenesis of the relevant amino Acid residues. We have expressed and purified nonconservative alanine (A) replacement mutants at the following amino Acid sequence locations in r-K2tPA:E17 (r-[K2tPA/E17A]), E75 (r-[K2tPA/E75A]), and D78 (r-[K2tPA/D78A]). More conservative E for D replacements were generated at the only other anionic (at neutral pH) amino Acids of r-[K2tPA], viz., D57 (r-[K2tPA/D57E]) and D59 (r-[K2tPA/D59E]). Each of these variant polypeptides was then utilized for binding investigations with a series of omega-amino Acids. No substantial differences were found in the binding constants (pH 8.0, 25 degrees C) for the ligands, 6-Aminohexanoic Acid (6-AHxA), 7-aminoheptanoic Acid (7-AHpA), L-lysine, and trans-(aminomethyl)cyclohexane-1-carboxylic Acid (AMCHA), among wild-type (wt) r-K2tPA, r-[K2tPA/E17A], r-[K2tPA/E75A], and r-[K2tPA/D78A]. On the other hand, dramatic effects on this same binding were observed in recombinant mutants with alterations at D57 and D59. In these cases, even with the most conservative replacements, i.e., r-[K2tPA/D57E] and r-[K2tPA/D59E], the Kd values for these ligands were increased approximately 3-6-fold and 18-85-fold, respectively. NMR analysis of these variants suggested that no substantial gross conformational changes occurred as a result of the mutations made, but some localized alterations in amino Acid microenvironments did take place.(ABSTRACT TRUNCATED AT 250 WORDS)

  • the cationic locus on the recombinant kringle 2 domain of tissue type plasminogen activator that stabilizes its interaction with omega amino Acids
    Biochemistry, 1992
    Co-Authors: V S De Serrano, Francis J. Castellino
    Abstract:

    : The properties of the cationic locus within the recombinant (r) kringle 2 domain (residues 180-261) of tissue-type plasminogen activator ([K2tPA]) that are responsible for stabilization of its interaction with the carboxylate moiety of omega-amino Acid ligands have been assessed by determination of the binding constants of several such ligands to a variety of r-[K2tPA] mutants obtained by oligonucleotide-directed mutagenesis. We have generated, expressed in Escherichia coli, and purified alanyl mutants of individual histidyl,lysyl, and arginyl residues of r-[K2tPA] and determined the dissociation constants of several omega-amino Acids, viz., 6-Aminohexanoic Acid (6-AHxA), 7-aminoheptanoic Acid (7-AHpA), L-lysine (L-Lys), and trans-(aminomethyl)cyclohexane-1-carboxylic Acid (AMCHA), to each of the r-[K2tPA] variants. We find that K33 plays the most significant role as a cationic partner of the complementary carboxylate group of these ligands. When K33 is altered to a variety of other amino Acids, the K33R mutant best stabilizes binding of all of these ligands. However, the r-K33L and r-K33F variants selectively interact with 7-AHpA almost as strongly (ca. 2-fold reduction in binding strength) as wild-type r-[K2tPA]. Increased polarity (K33Q) or a negative charge (K33E) at this sequence position significantly destabilizes binding of omega-amino Acids to the muteins. We also found that the r-K33E mutant and, to a lesser extent, the r-K33Q variant selectively interact with a new ligand, 1,6-diaminohexane. These observations show that the omega-amino Acid binding site of wtr-[K2tPA] could be redesigned to provide a new binding specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Miguel Llinas - One of the best experts on this subject based on the ideXlab platform.

  • ligand binding to the tissue type plasminogen activator kringle 2 domain structural characterization by 1h nmr
    Biochemistry, 1995
    Co-Authors: Inja L Byeon, Robert F Kelley, Michael G Mulkerrin, Seong Soo A An, Miguel Llinas
    Abstract:

    : Ligand binding to a recombinant human tissue-type plasminogen (tPA) kringle 2 domain has been characterized via 1H-NMR spectroscopy at 500 MHz. Seven omega-amino Acid ligands were investigated: L-Lys, 6-Aminohexanoic Acid (6AHA), 7-aminoheptanoic Acid (7AHA), trans-(aminomethyl)-cyclohexanecarboxylic Acid (AMCHA), p-(aminomethyl)benzoic Acid (PAMBA), p-(aminoethyl)benzoic Acid (PAEBA), and p-benzylaminesulfonic Acid (BASA). The interactions with two peptides containing a C-terminal lysyl residue, Tyr-Leu-Leu-Lys (YLLK) and Ala-Phe-Gln-Tyr-His-Ser-Lys (AFQYHSK), were also studied. The sequence AFQYHSK is found within the plasminogen N-terminal activation peptide while the tetrapeptide YLLK corresponds the 119-122 segment of the fibrinogen B beta-chain. Spectral comparison of ligand-free and ligand-containing kringle 2 samples leads to the conclusion that all the small ligands as well as the peptides' C-terminal lysyl residues interact with a common binding site in kringle 2. Two-dimensional spectra show that besides the Tyr36, Trp62, His64, Trp72, and Tyr74 aromatic rings, the Val35 and Asp55 aliphatic side chains also participate in ligand binding. Contact points with the ligands 6AHA and BASA were unambiguously identified from kringle 2-ligand nuclear Overhauser effects (NOEs). Overall, the ligand-induced chemical shifts and the intermolecular NOEs correlate remarkably well. Association constant (Ka) values for the kringle 2-ligand interactions were determined. Among the investigated ligands, BASA perturbs the kringle 2 spectrum the most and exhibits the highest affinity for kringle 2 (Ka approximately 233 mM-1). Of the two other aromatic ligands, PAEBA binds to kringle 2 less firmly (Ka = approximately 12 mM-1) than does the one-methylene group shorter analog PAMBA (Ka approximately 31 mM-1). By comparison, relative to 6AHA (Ka approximately 22 mM-1), the longer chain linear aliphatic ligand 7AHA interacts with kringle 2 with significantly higher affinity (Ka approximately 149 mM-1). By reference to the NMR-derived binding site structure, it is suggested that the higher affinity toward 7AHA may stem from (a) a relatively more favored ionic pairing between its carboxylate group and the LYs34 + Arg 69 side-chain cationic centers and (b) an enhanced interaction between the ligand hydrocarbon moiety and the kringle hydrophobic pocket, in particular with the Leu70 side chain. The latter is consistent with the relatively good affinity of kringle 2 for the cyclic hydrocarbon ligand AMCHA (Ka approximately 69 mM-1).(ABSTRACT TRUNCATED AT 400 WORDS)

  • solution structure of the tissue type plasminogen activator kringle 2 domain complexed to 6 aminohexanoic Acid an antifibrinolytic drug
    Journal of Molecular Biology, 1991
    Co-Authors: Inja L Byeon, Miguel Llinas
    Abstract:

    Abstract The solution structure of a recombinant tissue-type plasminogen activator kringle 2 domain, complexed with the antifibrinolytic drug 6-Aminohexanoic Acid (6-AHA) was determined via 1H nuclear magnetic resonance spectroscopy and dynamical simulated annealing calculations. The structure determination is based on 610 intramolecular kringle 2 and 14 intermolecular kringle 2-6-AHA interproton distance restraints, as well as on 82 torsion angle restraints. Three sets of simulated annealing structures were computed from three different classes of starting structures: (1) random conformations devoid of disulfide bridges; (2) random conformations that contain correct disulfide bonds; and (3) a folded conformation modeled after the homologous prothrombin kringle 1 X-ray crystallographic structure. All three sets of structures are well defined, with averaged atomic root-meansquare deviations between individual structures and mean set structures of 0·77, 0·99 and 0·70 A for backbone atoms, and 1·36, 1·55 and 1·41 A for all atoms, respectively. Kringle 2 is an oblate ellipsoid with overall dimensions of approximately 34 A × 30 A × 17 A. It exhibits a compact globular conformation characterized by a number of turns and loop elements as well as by one right-handed α-helix and five (1 extended and 4 rudimentary) antiparallel β-sheets. The extended β-sheet exhibits a right-handed twist. Close van der Waals' contacts between the Cys22-Cys63 and Cys51-Cys75 disulfide bridges and the central hydrophobic core composed of the Trp25, Leu46, His48a and Trp62 sidechains are among the distinguishing features of the kringle 2 fold. The binding site for 6-AHA appears as a rather exposed cleft with a negatively charged locus defined by the Asp55 and Asp57 side-chains, and with an aromatic pocket structured by the Tyr36, Trp62, His64 and Trp72 side-chains. The Trp62 and His64 rings line the back surface of the pocket, while the Tyr36 and Trp72 rings confine it from two sides. The Trp62 and Trp72 indole rings conform a V-shaped groove. The methyl groups of Va135 also contribute lipophilic character to the ligand-interacting surface. It is suggested that the positively charged side-chains of Lys34 and, potentially, Arg69 may favor interactions with the carboxylate group of the ligand. The Trp25 and Tyr74 aromatic rings, although conserved elements of the binding site structure, seem not to undergo direct contacts with the ligand.

Inja L Byeon - One of the best experts on this subject based on the ideXlab platform.

  • ligand binding to the tissue type plasminogen activator kringle 2 domain structural characterization by 1h nmr
    Biochemistry, 1995
    Co-Authors: Inja L Byeon, Robert F Kelley, Michael G Mulkerrin, Seong Soo A An, Miguel Llinas
    Abstract:

    : Ligand binding to a recombinant human tissue-type plasminogen (tPA) kringle 2 domain has been characterized via 1H-NMR spectroscopy at 500 MHz. Seven omega-amino Acid ligands were investigated: L-Lys, 6-Aminohexanoic Acid (6AHA), 7-aminoheptanoic Acid (7AHA), trans-(aminomethyl)-cyclohexanecarboxylic Acid (AMCHA), p-(aminomethyl)benzoic Acid (PAMBA), p-(aminoethyl)benzoic Acid (PAEBA), and p-benzylaminesulfonic Acid (BASA). The interactions with two peptides containing a C-terminal lysyl residue, Tyr-Leu-Leu-Lys (YLLK) and Ala-Phe-Gln-Tyr-His-Ser-Lys (AFQYHSK), were also studied. The sequence AFQYHSK is found within the plasminogen N-terminal activation peptide while the tetrapeptide YLLK corresponds the 119-122 segment of the fibrinogen B beta-chain. Spectral comparison of ligand-free and ligand-containing kringle 2 samples leads to the conclusion that all the small ligands as well as the peptides' C-terminal lysyl residues interact with a common binding site in kringle 2. Two-dimensional spectra show that besides the Tyr36, Trp62, His64, Trp72, and Tyr74 aromatic rings, the Val35 and Asp55 aliphatic side chains also participate in ligand binding. Contact points with the ligands 6AHA and BASA were unambiguously identified from kringle 2-ligand nuclear Overhauser effects (NOEs). Overall, the ligand-induced chemical shifts and the intermolecular NOEs correlate remarkably well. Association constant (Ka) values for the kringle 2-ligand interactions were determined. Among the investigated ligands, BASA perturbs the kringle 2 spectrum the most and exhibits the highest affinity for kringle 2 (Ka approximately 233 mM-1). Of the two other aromatic ligands, PAEBA binds to kringle 2 less firmly (Ka = approximately 12 mM-1) than does the one-methylene group shorter analog PAMBA (Ka approximately 31 mM-1). By comparison, relative to 6AHA (Ka approximately 22 mM-1), the longer chain linear aliphatic ligand 7AHA interacts with kringle 2 with significantly higher affinity (Ka approximately 149 mM-1). By reference to the NMR-derived binding site structure, it is suggested that the higher affinity toward 7AHA may stem from (a) a relatively more favored ionic pairing between its carboxylate group and the LYs34 + Arg 69 side-chain cationic centers and (b) an enhanced interaction between the ligand hydrocarbon moiety and the kringle hydrophobic pocket, in particular with the Leu70 side chain. The latter is consistent with the relatively good affinity of kringle 2 for the cyclic hydrocarbon ligand AMCHA (Ka approximately 69 mM-1).(ABSTRACT TRUNCATED AT 400 WORDS)

  • solution structure of the tissue type plasminogen activator kringle 2 domain complexed to 6 aminohexanoic Acid an antifibrinolytic drug
    Journal of Molecular Biology, 1991
    Co-Authors: Inja L Byeon, Miguel Llinas
    Abstract:

    Abstract The solution structure of a recombinant tissue-type plasminogen activator kringle 2 domain, complexed with the antifibrinolytic drug 6-Aminohexanoic Acid (6-AHA) was determined via 1H nuclear magnetic resonance spectroscopy and dynamical simulated annealing calculations. The structure determination is based on 610 intramolecular kringle 2 and 14 intermolecular kringle 2-6-AHA interproton distance restraints, as well as on 82 torsion angle restraints. Three sets of simulated annealing structures were computed from three different classes of starting structures: (1) random conformations devoid of disulfide bridges; (2) random conformations that contain correct disulfide bonds; and (3) a folded conformation modeled after the homologous prothrombin kringle 1 X-ray crystallographic structure. All three sets of structures are well defined, with averaged atomic root-meansquare deviations between individual structures and mean set structures of 0·77, 0·99 and 0·70 A for backbone atoms, and 1·36, 1·55 and 1·41 A for all atoms, respectively. Kringle 2 is an oblate ellipsoid with overall dimensions of approximately 34 A × 30 A × 17 A. It exhibits a compact globular conformation characterized by a number of turns and loop elements as well as by one right-handed α-helix and five (1 extended and 4 rudimentary) antiparallel β-sheets. The extended β-sheet exhibits a right-handed twist. Close van der Waals' contacts between the Cys22-Cys63 and Cys51-Cys75 disulfide bridges and the central hydrophobic core composed of the Trp25, Leu46, His48a and Trp62 sidechains are among the distinguishing features of the kringle 2 fold. The binding site for 6-AHA appears as a rather exposed cleft with a negatively charged locus defined by the Asp55 and Asp57 side-chains, and with an aromatic pocket structured by the Tyr36, Trp62, His64 and Trp72 side-chains. The Trp62 and His64 rings line the back surface of the pocket, while the Tyr36 and Trp72 rings confine it from two sides. The Trp62 and Trp72 indole rings conform a V-shaped groove. The methyl groups of Va135 also contribute lipophilic character to the ligand-interacting surface. It is suggested that the positively charged side-chains of Lys34 and, potentially, Arg69 may favor interactions with the carboxylate group of the ligand. The Trp25 and Tyr74 aromatic rings, although conserved elements of the binding site structure, seem not to undergo direct contacts with the ligand.

C N Rao - One of the best experts on this subject based on the ideXlab platform.

  • matrix localization of tissue factor pathway inhibitor 2 matrix associated serine protease inhibitor tfpi 2 mspi involves arginine mediated ionic interactions with heparin and dermatan sulfate heparin accelerates the activity of tfpi 2 mspi toward pl
    Archives of Biochemistry and Biophysics, 1999
    Co-Authors: Yueying Liu, Sharon M Stack, Sanjani S Lakka, Azim J Khan, David T Woodley, Jasti S Rao, C N Rao
    Abstract:

    Abstract Human tissue factor pathway inhibitor-2 (TFPI-2)/matrix-associated serine protease inhibitor (MSPI), a Kunitz-type serine protease inhibitor, inhibits plasmin, trypsin, chymotrypsin, plasma kallikrein, cathepsin G, and factor VIIa–tissue factor complex. The mature protein has a molecular mass of 32–33 kDa, but exists in vivo as two smaller, underglycosylated species of 31 and 27 kDa. TFPI-2/MSPI triplet is synthesized and secreted by a variety of cell types that include epithelial, endothelial, and mesenchymal cells. Because the majority (75–90%) of TFPI-2/MSPI is associated with the extracellular matrix (ECM), we examined which components of the ECM bind TFPI-2/MSPI. We found that TFPI-2/MSPI bound specifically to heparin and dermatan sulfate. Interaction of these two glycosaminoglycans (GAGs) with TFPI-2/MSPI involved one or more common protein domains, as evidenced by cross-competition experiments. However, binding affinity for TFPI-2/MSPI with heparin was 250–300 times greater than that for TFPI-2/MSPI with dermatan sulfate. Binding of TFPI-2/MSPI to GAGs was inhibited by NaCl or arginine but not by glucose, mannose, galactose, 6-Aminohexanoic Acid, or urea, suggesting that arginine-mediated ionic interactions participate in the GAG binding of TFPI-2/MSPI. This supposition was supported by the observation that only NaCl or arginine could elute the TFPI-2/MSPI protein triplet from an ECM derived from human dermal fibroblasts. Reduced TFPI-2/MSPI did not bind to heparin, suggesting that proper disulfide pairings and conformation are essential for matrix binding. To determine whether heparin modulates the activity of TFPI-2/MSPI, we determined the rate of inhibition of plasmin by the inhibitor with and without heparin and found that TFPI-2/MSPI is more active in the presence of heparin. Collectively, our results demonstrate that conformation-dependent arginine-mediated ionic interactions are responsible for the TFPI-2/MSPI triplet binding to fibroblast ECM, heparin, and dermatan sulfate and that heparin augmented the rate of inhibition of plasmin by TFPI-2/MSPI.