Solution Structure of the Epsilon-Aminohexanoic Acid Complex of Human Plasminogen Kringle1
Rejante, M.R. & Llinás, M. Eur.J.Biochem., 221, 939 (1994)
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.
Plasminogen is a precursor of the anticoagulant protein plasmin. Plasmin, a serine protease, activates the dissociation of the fibrin matrix of blood clots. A single human plasminogen peptide chain consists of 791 amino acid residues and two carbohydrates attached at the Asn-289 and Thr-346. With the presence of disulfide bonds, plasminogen's structure is divided into a contiguous series of five homologous regions called kringles. These modules are named as such for their resemblance of the danish pastry: danish kringle. Human plasminogen consists of an amino-terminal peptide, five kringle modules and a protease domain. Plasminogen is converted to plasmin by cleavage of Lys-77 releasing the N-terminal peptide. Activator proteins cleavage of the peptide bond at Arg-561 then follows. A two chain molecule is formed in which the heavy chain containing the five modules remains linked to the light chain where the catalytic domain is found through 2 disulfide bonds.
Each kringle module is comprised of about 78 residues which are very homologous in sequence. Kringles are also present in a number of blood coagulation proteins and serine proteases including prothrombin, tissue-type plasminogen activators, urokinase plasminogen activator, Factor X and etc. In addition, these modules can also be found in hepatocyte growth factors which are involved in the increase in motility of various epithelial and endothelial cells and the proliferation of primary hepatocytes. The presence of kringles in these different proteins suggest that these enzymes might share a common primitive ancestor during the course of evolution.
Primary Structure of Human Plasminogen
(click to enlarge picture)