Lysine-50 is a likely site for anchoring the plasminogen N-terminal peptide to lysine-binding kringles

Interactions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N-terminal Glu1-Lys77 peptide (NTP) have been investigated via 1H-NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant K(a) < 0.05 mM-1). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative N(α)-acetyl-L- lysine-methyl ester (K(a) ~ 0.2 mM-1), peptides encompassing Lys50 consistently show higher K(a) values. Among the investigated linear peptides, N(α)-acetyl-Ala-Phe-Tyr-His-Ser-Ser-Lys50-Glu-Gln-NH2 (AcAFYHSK50EQ-NH2) exhibits the strongest interaction with K4 (K(a) ~ 1.4 mM-1), followed by AcYHSK50EQ-NH2 (K(a) ~ 0.9 mM-1). Relative to the wild-type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 → Ser mutation was introduced (→ AcYHSS50EQ-NH2), binding was abolished. The Ile27-Ile56 construct (L-NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant Pgn K1 (rK1) and K2 (rK2) modules, and of Pgn fragments encompassing the intact K4 and K5 domains, for binding L-NTP, was investigated. We find that L-NTP interacts with rK1, rK2, K4, and K5-all lysine-binding kringles-in a fashion that closely mimics what has been observed for the Glu1-HSer57 N-terminal fragment of Pgn (CB-NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L-NTP and magnitudes of kringle-L-NTP binding affinities (rK1, K(a) ~ 4.3 mM-1; rK2, K(a) ~ 3.7 mM-1; K4, K(a) ~ 6.4 mM-1; and K5, K(a) ~ 2.1 mM-1) are essentially the same as for the corresponding kringle-CB-NTP pairs. Molecular modeling studies suggest that the Glu39-Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent-exposed kringle LBS surface.