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 (P gn) is followed by a tandem array of five kringle (K) structures of si milar 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 f ragment (CB-NTP) isolated. In addition, the Ile27-Ile56 peptide (L-NTP ) that spans the doubly S-S bridged loop segment of NTP was synthesize d. Pgn kringles were generated either by proteolytic fragmentation of Pgn (K4, K5) or via recombinant gene expression (rK1, rK2, and rK3). I nteractions of CB-NTP with each of the Pgn kringles were monitored by H-1-NMR at 500 MHz and values for the equilibrium association constant s (K-a) determined: rK1, K-a similar to 4.6 mM(-1); rK2, K-a similar t o 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 stro ngly than with Na-acetyl-L-lysine methyl ester (K-a < 0.6 mM(-1)), whi ch reveals specificity for the NTP. In contrast, CB-NTP does not measu rably 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 Glu 1-Ile27 segment of CB-NTP were computed via restrained dynamic simulat ed annealing/energy minimization (SA/EM) protocols. Conformational mod els of CB-NTP were generated by joining the two (sub)structures follow ed by a round of constrained SA/EM. Helical turns are indicated for se gments 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 Lys 50 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.