Solution structure and dynamics of the plasminogen kringle 2-AMCHA complex: 3(1)-helix in homologous domains

The kringle 2 (K2) module of human plasminogen (Pgn) binds L-lysine and ana logous zwitterionic compounds, such as the antifibrinolytic agent trans-(am inomethyl) cyclohexanecarboxylic acid (AMCHA). Far-UV CD and NMR spectra re veal little conformational change in K2 upon ligand binding. However, retar ded H-1-H-2 isotope exchange kinetics induced by AMCHA indicate stabilizati on of the K2 conformation by the ligand. Assessment of secondary structure content from CD spectra yields similar to 26% beta-strand, similar to 13% b eta-turn, similar to 15% 3(1)-helix, and similar to 6% 3(10)-helix. The NMR solution conformation of the K2 domain complexed to AMCHA has been determi ned [heavy atom rmsd = 0.49 +/- 0.09 Angstrom (backbone) and 1.02 +/- 0.08 Angstrom tall)]. The K2 molecule has overall dimensions of similar to 34.5 Angstrom x similar to 33.4 Angstrom x similar to 22.7 Angstrom. Analogous w ith the polypeptide outline of homologous domains, K2 contains three short antiparallel beta-sheets (paired strands 15-16/20-21, 24-25/48-49, and 62-6 4/72-74) and four defined beta-turns (residues 6-9, 16-19, 53-56, and 67-70 ). Consistent with the CD analysis, albeit novel in the context of kringle folding, the NMR structure reveals an unpaired beta strand structured by re sidues 30-32, a turn of 3(10)-helix comprising residues 38-41, and a 3(1)-h elix for residues 21-24 and 74-79. We also identify alignable 3(1)-helices in previously reported homologous kringle structures. Rather high order par ameter S-2 values ([S-2] similar to 0.85 +/- 0.04) characterize the K2 back bone dynamics. The lowest flexibility is observed for the two inner loop se gments of residues 51-63 and 63-75 ([S-2] similar to 0.86-0.87 +/- 0.03). O verhauser connectivities reveal close hydrophobic contacts of the ligand ri ng with side chains of Tyr(36), Trp(62), Phe(64) Trp(72), and Leu(74). In m ost K2 structures, the N atom of AMCHA places itself similar to 3.9 and sim ilar to 4.4 Angstrom from the anionic groups of Glu(57) and Asp(55), respec tively, while its carboxylate group, H-bonded to the Tyr36 Side chain OHeta , ion-pairs the Arg(71) guanidinium group. Consistent with the preference o f K2 for binding 5-aminopentanoic acid over 6-aminohexanoic acid, the posit ions of the ionic centers within the K2 binding site approach each other si milar to 1 closer relative to what is observed in lysine binding sites of h omologous Pgn modules.