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

Abstract

Interactions between the kringle 4 (K4) domain of human plasminogen (Pgn) and segments of the N-terminal GlulLys77 peptide (NTP) have been investigated via '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, < 0.05 mM"). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Ne-acetyl-L-lysine-methyl ester (K, -0.2 mM-'), peptides encompassing Lys50 consistently show higher K, values. Among the investigated linear peptides, N"-acetyl-Ala-Phe-Tyr-His-Ser-Ser-Lys50-Glu-Gln-NH2 (AcAFYHSKSOEQ-NH,) exhibits the strongest interaction with K4 (KO -1.4 mM"), followed by AcYHSKSOEQ-NH2 (K, -0.9 mM"). Relative to the wild-type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 + Ser mutation was introduced (* AcYHSSSOEQ-NH,), 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, -4.3 mM" ; rK2, K, -3.7 mM" ; K4, K, -6.4 mM" ; and K5, K, -2.1 mM-' ) 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.

Gelatin-binding Region of Human Matrix Metalloproteinase-2

“…Thus, by providing an anchoring site for the prodomain, COL-3 would stabilize the pro-MMP-2 in a compact conformation; in contrast, the main role of COL-2 may be that of promoting the binding interaction with the gelatin substrate. This is reminiscent of what has been observed for plasminogen, the proenzyme of plasmin, where the five kringle domains, which exhibit various degrees of affinity for lysinecontaining peptides (putative anchoring sites in the plasminogen-fibrin interaction), also differ in their affinities for the plasminogen N-terminal peptide, suggesting that they may selectively regulate the compact folding of the macromolecule (47,57). Following activation to plasmin, the N-terminal peptide is autolytically cleaved off, causing plasminogen to assume an "open" conformation (Ref.…”

A new series of the SMTP plasminogen modulator with a phenylglycine-based side chain

“…1 Plasminogen adopts tight conformation due to intramolecular binding of Lys 50 and/or Lys 62 to the lysinebinding site in the fifth kringle domain. 3,4 The tight conformation renders plasminogen less sensitive to activation by plasminogen activators. Plasminogen binding to fibrin or cellular receptors allows relaxation of plasminogen conformation, enabling efficient activation.…”

Expression and characterization of recombinant human micro-plasminogen

“…The kringle 1-5 domain of Glu-plasminogen participates an intramolecular interaction with Lys 50 and/or Lys 62 by bringing the N-terminal peptide domain in close contact with the protease domain and leads to a very tight spiral structure of Glu-plasminogen (Cockell et al 1998;An et al 1998). This structure was thought to shield the activation cleavage site (Arg 561 -Val 562 ) from easy access by plasminogen activator (Horrevoets et al 1995).…”