Solution Structure and Dynamics of the Plasminogen Kringle 2−AMCHA Complex:  3₁-Helix in Homologous Domains^†,‡

The kringle 2 (K2) module of human plasminogen (Pgn) binds l-lysine and analogous zwitterionic compounds, such as the antifibrinolytic agent trans-(aminomethyl)cyclohexanecarboxylic acid (AMCHA). Far-UV CD and NMR spectra reveal little conformational change in K2 upon ligand binding. However, retarded ¹H−²H isotope exchange kinetics induced by AMCHA indicate stabilization of the K2 conformation by the ligand. Assessment of secondary structure content from CD spectra yields ∼26% β-strand, ∼13% β-turn, ∼15% 3₁-helix, and ∼6% 3₁₀-helix. The NMR solution conformation of the K2 domain complexed to AMCHA has been determined [heavy atom rmsd = 0.49 ± 0.09 Å (backbone) and 1.02 ± 0.08 Å (all)]. The K2 molecule has overall dimensions of ∼34.5 Å × ∼33.4 Å × ∼22.7 Å. Analogous with the polypeptide outline of homologous domains, K2 contains three short antiparallel β-sheets (paired strands 15−16/20−21, 24−25/48−49, and 62−64/72−74) and four defined β-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 β-strand structured by residues 30−32, a turn of 3₁₀-helix comprising residues 38−41, and a 3₁-helix for residues 21−24 and 74−79. We also identify alignable 3₁-helices in previously reported homologous kringle structures. Rather high order parameter S² values (〈S²〉 ∼ 0.85 ± 0.04) characterize the K2 backbone dynamics. The lowest flexibility is observed for the two inner loop segments of residues 51−63 and 63−75 (〈S²〉 ∼ 0.86−0.87 ± 0.03). Overhauser connectivities reveal close hydrophobic contacts of the ligand ring with side chains of Tyr³⁶, Trp⁶², Phe⁶⁴, Trp⁷², and Leu⁷⁴. In most K2 structures, the N atom of AMCHA places itself ∼3.9 and ∼4.4 Å from the anionic groups of Glu⁵⁷ and Asp⁵⁵, respectively, while its carboxylate group, H-bonded to the Tyr³⁶ side chain OH^η, ion-pairs the Arg⁷¹ guanidinium group. Consistent with the preference of K2 for binding 5-aminopentanoic acid over 6-aminohexanoic acid, the positions of the ionic centers within the K2 binding site approach each other ∼1 Å closer relative to what is observed in lysine binding sites of homologous Pgn modules.