KRINGLE SOLUTION STRUCTURES VIA NMR - 2-DIMENSIONAL H-1-NMR ANALYSIS OF HORSE PLASMINOGEN KRINGLE-4

The kringle 4 domain of equine plasminogen (ePgn/K4), a close variant of the human homolog (hPgn/K4), contains residues, such as Trp(32), which also appear in human apolipoprotein(a) kringle 4-type modules. The ePgn/K4 was investigated as a complex with E-aminocaproic acid, an antifibrinolytic drug, by two-dimensional H-1-NMR spectroscopy at 500 MHz. Secondary structure elements were recognized from sequential medium and long-range dipolar (proton Overhauser) interactions, as well as from the identification of resonances originating from backbone amide protons with slow H-1-H-2 exchange in (H2O)-H-2. Antiparallel beta-sheets, consisting of strands 52-53, 61-65 and 71-75, were identified. Additionally, the segments 14-16 and 20-22 were found to assume characteristic interstrand antiparallel (beta-sheet-like) H-bond pairing. Four type I turns could be identified in strands 6-9, 16-19, 24-27 and 67-70. Ten structures were generated using distance geometry methods, followed by dynamic simulated annealing calculations. The root mean squares deviation of the distances was 2.79 Angstrom for all atoms and 1.81 Angstrom for backbone atoms only. Hydrogen bridges, involving side chain hydroxyl groups, were identified for Thr(16) and Thr(65). AS observed for the hPgn/K4, the three-dimensional structure of the ePgn/K4 is mainly defined by two antiparallel beta-sheets, 14-16/20-22 and 62-66/71-75, which are oriented perpendicular to each other. Adjacent to these is a hydrophobic pocket, formed by Trp(62), Tyr(64), Trp(72) and Phe(74), whose side chains contribute a lipophilic component to the exposed lysine binding site surface. In contrast to the Trp(25), Trp(62) and Trp(72) residues, conserved in the human and equine homologs, the spectrum of the Trp(32) side chain reveals an unrestrained, solvent-exposed indole ring.