Solution structure of the ɛ‐aminohexanoic acid complex of human plasminogen kringle 1

Abstract

The solution structure of the human plasminogen kringle 1 domain complexed to the antifibrinolytic drug 6-aminohexanoic acid (epsilon Ahx) was obtained on the basis of 1H-NMR spectroscopic data and dynamical simulated annealing calculations. Two sets of structures were derived starting from (a) random coil conformations and (b) the (mutated) crystallographic structure of the homologous prothrombin kringle 1. The two sets display essentially the same backbone folding (pairwise root-mean-square deviation, 0.15 nm) indicating that, regardless of the initial structure, the data is sufficient to locate a conformation corresponding to an essentially unique energy minimum. The conformations of residues connected to prolines were localized to energetically preferred regions of the Ramachandran map. The Pro30 peptide bond is proposed to be cis. The ligand-binding site of the kringle 1 is a shallow cavity composed of Pro33, Phe36, Trp62, Tyr64, Tyr72 and Tyr74. Doubly charged anionic and cationic centers configured by the side chains of Asp55 and Asp57, and Arg34 and Arg71, respectively, contribute to anchoring the zwitterionic epsilon Ahx molecule at the binding site. The ligand exhibits closer contacts with the kringle anionic centers (approximately 0.35 nm average O...H distance between the Asp55/Asp57 carboxylate and ligand amino groups) than with the cationic ones (approximately 0.52 nm closest O...H distances between the ligand carboxylate and the Arg34/Arg71 guanidino groups). The epsilon Ahx hydrocarbon chain rests flanked by Pro33, Tyr64, Tyr72 and Tyr74 on one side and Phe36 on the other. Dipolar (Overhauser) connectivities indicate that the ligand aliphatic moiety establishes close contacts with the Phe36 and Trp62 aromatic rings. The computed structure suggests that the epsilon Ahx molecule adopts a kinked conformation when complexed to kringle 1, effectively shortening its dipole length to approximately 0.65 nm.

1H‐NMR assignments and secondary structure of human plasminogen kringle 1

“…Thus, it is suggested that the hairpin turn connecting the two antiparallel strands differs among the kringles with a type I turn spanning residues 67-70 in the case of PgdK4. Structure calculations [41] show a similar backbone conformation for PgdK1 in this region (Fig. 7H).…”

“…A bifurcated hydrogen bond involving the Thrl6 carbonyl (Fig. 6 A) is suggested by complete structure calculations [41]. The stretch Lys24-Trp25-Ser26-Ser27 delineates a type I or 11' turn [62].…”

“…The turn formed by this Pro was classified as open, not reverse [31]. Structure calculations [41] on the Pgn/Kl show that better convergence is obtained when Pro30 is constrained to the cis conformation. On the rare occasions when cis prolines are found, they usually occupy the third position of a type VIa or VIh turn [68].…”

“…The proximity of Ser26 to Arg34, indicated by a weak amide-to-amide NOE, suggests that the intervening residues form a turn or compact loop. Similarly, the stretch connecting the two short, antiparallel p strands formed by Trp62-Cys63-Tyr64-Thr65 and Arg71-Tyr72-Asp73-Tyr74 must be involved in a turn or loop [41] (Figs 6B and 7H). A weak du,(i,i+4) NOE between Asp67 and Arg71 suggests that a turn may be formed by the intervening residues (Fig.…”

“…From the analysis, and by reference to PgdK4 and tPA/K2, it is also suggested that, although the main features of the secondary structure are conserved among the three homologs, there are variations in a number of the structural elements. The studies presented here constitute the ground work for a complete solution structure determination reported in the following paper [41].…”