Kringle solution structures via NMR: two-dimensional ¹H-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 Trp³², which also appear in human apolipoprotein(a) kringle 4-type modules. The ePgn/K4 was investigated as a complex with ε{lunate}-aminocaproic acid, an antifibrinolytic drug, by two-dimensional ¹H-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 ¹H-²H exchange in ²H₂O. Antiparallel β-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 (β-sheet-like) Hbond 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 Å for all atoms and 1.81 Å for backbone atoms only. Hydrogen bridges, involving side chain hydroxyl groups, were identified for Thr¹⁶ and Thr⁶⁵. As observed for the hPgn/K4, the three-dimensional structure of the ePgn/K4 is mainly defined by two antiparallel β-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 Trp⁶², Tyr⁶⁴, Trp⁷² and Phe⁷⁴, whose side chains contribute a lipophilic component to the exposed lysine binding site surface. In contrast to the Trp²⁵, Trp⁶² and Trp⁷² residues, conserved in the human and equine homologs, the spectrum of the Trp³² side chain reveals an unrestrained, solvent-exposed indole ring.