Structural/functional properties of the Glu1-HSer57 N-terminal fragment of human plasminogen:: Conformational characterization and interaction with kringle domains

The Glu1-Val79 N-terminal peptide (NTP) domain of human plasminogen (Pgn) is followed by a tandem array of five kringle (K) structures of similar to 9 kDa each. K1, K2, K4, and K5 contain each a lysine-binding site (LBS). Pgn was cleaved with CNBr and the Glu1-HSer57 N-terminal fragment (CB-NTP) isolated. In addition, the Ile27-Ile56 peptide (L-NTP) that spans the doubly S-S bridged loop segment of NTP was synthesized. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). Interactions of CB-NTP with each of the Pgn kringles were monitored by H-1-NMR at 500 MHz and values for the equilibrium association constants (K-a) determined: rK1, K-a similar to 4.6 mM(-1); rK2, K-a similar to 3.3 mM(-1); K4, K-a similar to 6.2 mM(-1); K5, K-a similar to 2.3 mM(-1). Thus, the lysine-binding kringles interact with CB-NTP more strongly than with Na-acetyl-L-lysine methyl ester (K-a < 0.6 mM(-1)), which reveals specificity for the NTP. In contrast, CB-NTP does not measurably interact with rK3, which is devoid of a LBS. CB-NTP and L-NTP H-1-NMR spectra were assigned and interproton distances estimated from H-1-H-1 Overhauser (NOESY) experiments. Structures of L-NTP and the Glu1-Ile27 segment of CB-NTP were computed via restrained dynamic simulated annealing/energy minimization (SA/EM) protocols. Conformational models of CB-NTP were generated by joining the two (sub)structures followed by a round of constrained SA/EM. Helical turns are indicated for segments 6-9, 12-16, 28-30, and 45-48. Within the Cys34-Cys42 loop of L-NTP, the structure of the Glu-Glu-Asp-Glu-Glu39 segment appears to be relatively less defined, as is the case for the stretch containing Lys50 within the Cys42-Cys54 segment, consistent with the latter possibly interacting with kringle domains in intact Glu1-Pgn. Overall, the CB-NTP and L-NTP fragments are of low regular secondary structure content-as indicated by W-CD spectra-and exhibit fast amide H-1-H-2 exchange in (H2O)-H-2, suggestive of high flexibility.