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 Glul‐Lys77 peptide (NTP) have been investigated via 1 H‐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‐NH 2 (AcAFYHSKSOEQ‐NH 2 ) exhibits the strongest interaction with K4 (K a ˜ 1.4 mM −1 ), followed by AcYHSKSOEQ‐NH 2 (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 (⇒ AcYHSSSOEQ‐NH 2 ), 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 (rKI) 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 Glul‐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 2 ˜ 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 GIu39‐Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent‐exposed kringle LBS surface.