Plasminogen Kringle Research Articles

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 α-acetyl- l-lysine (AcLys) and ϵ-aminocaproic acid (ϵACA) 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 (His 31), Trp-I (Trp 25?), Trp-II (Trp 62?), Trp-III (Trp 72), Tyr-II (Tyr 50), and Phe 64 ring signals. BASA tends to induce larger shifts than elicited by the aliphatic ligands, most noticeably on Trp-II and on Trp 72, 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 (Tyr 50), Tyr-III (Tyr 41), and Tyr-IV (Tyr 74) over a wide pH range and lowers the pK a ∗ of His 31 from ~4.8 to ~4.6. His-III (His 33) responds to BASA and AMCHA but is relatively insensitive to the linear ligands. His 33 carries a sterically shielded side chain which, in conjunction with Leu 46, Trp-I, Tyr 50, and Tyr 74, 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 ϵACA. It is proposed that the lysine-binding site is mostly supported by the loop that extends from Cys 51 through Cys 71 and that aromatic residues, which include Trp-II, Trp 72, and Phe 64, play a major role in interacting with the nonpolar segment of the ligand molecule. The binding site also encompasses Tyr 50, Tyr 74, His 31, and His 33 although it is not clear the extent to which these residues interact directly with the ligand.

Chemical modification and nuclear magnetic resonance studies on human plasminogen kringle 4. Assignment of tyrosine and histidine resonances to specific residues in the sequence.

Modification of kringle 4 with tetranitromethane leads to the selective nitration of tyrosine 40 but on prolonged incubation with reagent, reaction of tyrosine 49 is also observed. Nitration of tyrosines 40 and 49 had no influence on the lysine-Sepharose affinity of kringle 4, indicating that these residues are not important for the functional integrity of the ligand-binding site. Comparison of the NMR spectra of native kringle 4 with those of kringle 4 in which tyrosine 40 or tyrosines 40 and 49 are nitrated permitted the identification of the resonances of these residues. These NMR studies also showed that the chemical modifications caused little perturbation of the three-dimensional structure of the protein. Cross-linking of lysine 35 and tyrosine 40 with 1,3-difluoro-4,6-dinitrobenzene demonstrates that in the kringle-fold the reactive epsilon-amino and phenolic groups of these residues can approach each other to a distance of 0.5 nm. NMR spectra of this kringle 4 species also confirmed the assignment of the resonances to tyrosine 40. NMR spectra of a kringle 4 derivative in which the disulphide bridge between cysteines 1 and 79 has been broken by selective reduction and alkylation showed that the core structure of the kringle-fold and the lysine-binding site are unaltered by this modification. This observation is in agreement with earlier results which showed that the lysine-Sepharose affinity of kringle 4 is not affected by reduction and alkylation of this disulphide bridge. Comparison of the NMR spectra of native and disulphide-cleaved kringle 4 aided in the assignment of resonances to residues adjacent to the site of modification (tyrosine 2 and histidine 3) and permitted the tentative assignment of the resonances of tyrosines 9 and 73.

Characterization of a complementary deoxyribonucleic acid coding for human and bovine plasminogen

A cDNA library was constructed in pBR322 from bovine liver mRNA that was enriched for plasminogen mRNA by polysome immunoprecipitation. A 32P-labeled single-stranded cDNA was then prepared from the enriched bovine mRNA and employed as a probe to screen the cDNA library. The screening was carried out by testing for clones that protect the hybridized 32P-labeled cDNA from S1 nuclease digestion. The longest clone that was found was 581 base pairs in length and coded for the C-terminal 107 amino acids of bovine plasminogen, a 3' noncoding region of 246 nucleotides and a poly(A) tail. The bovine cDNA clone was then used as a probe to screen a human liver cDNA library of 18 000 recombinants. Six isolates were found to contain human plasminogen sequences. The longest clone consisted of 1851 base pairs corresponding to amino acid residues 272-790, followed by a 3' noncoding region of 227 base pairs and a poly(A) tail. Restriction fragments of the human cDNA were then used as probes to screen a human genomic DNA library present in a Charon 4A lambda phage library. Approximately 50 isolates from 10(6) recombinants were identified that hybridized to varying degrees with the cDNA probe. Among these, 10 corresponding to the gene for human plasminogen have been analyzed, and 3 that overlap have been shown to extend from kringle 3 through the 3' noncoding region of the gene. A 160 base pair exon with flanking splice junctions was then characterized and shown to encode for the first half of plasminogen kringle 4, including amino acid residues 346-399.

Folding autonomy of the kringle 4 fragment of human plasminogen.

Kringle 4, an 88-residue plasminogen fragment carrying a lysine-binding site, loses its affinity for lysine-Sepharose upon reductive cleavage of its disulfide bridges. Aerobic incubation of the reduced, denatured fragment results in the rapid restoration of the disulfide bonds with concomitant recovery of lysine-Sepharose affinity. The ability of the unfolded fragment to regain its native conformation suggests that the kringle structure is an autonomous folding domain. During refolding of kringle 4 the native disulfide bonds, (formula; see text) and (formula; see text), appears first. The folding intermediate possessing these two disulfide bridges already binds to lysine-Sepharose, indicating that the third native bridge, which in native kringle 4 connects residues Cys1 and Cys79, is not essential for the maintenance of the biologically active conformation of kringle 4. Comparison of the sequences of human prothrombin, urokinase, and plasminogen kringles revealed that the residues surrounding the (formula; see text) and (formula; see text) bridges constitute the most conservative segments of kringles, whereas the residues neighboring the (formula; see text) bridge are not highly conserved. We propose that conservation of various residues in the different kringles reflects their importance for the folding autonomy of kringles.