Abstract
One of the key proteins involve in clot lysis in vertebrate organisms is plasminogen (Glu1-Pg). It is the circulating zymogen of the serine protease plasmin (Pm). Generation of the active enzyme requires cleavage of the Arg561-Val562 bond and proteolytic removal of the 77-residue, N-terminal activation peptide (AP). Binding of Lys and Lys-analogs by Pg/Pm is mediated by kringle domains (K), and they determine the interaction of Pg/Pm with substrates, receptors, activators and inhibitors. Activation rate of Pg is highly dependent on its conformation and it has been shown that the zymogen can assume at least two major conformational states; closed and open. The former is resistant to activation and is detected in the presence of Cl-, and the latter is more easily activated and is observed in the presence of Lys and Lys analogs, such as 6-amino hexanoic acid (6AHA). Pg lacking the AP (Lys78-Pg) exists in the open conformation, suggesting that intramolecular interactions between the AP and the kringle domains of Glu1-Pg induce the closed form of the zymogen. To assess the contributions of specific domains on the maintenance of the closed form, we have generated a panel of recombinant Pg truncation mutants, which include APK1-3 (residues 1-338 of Pg), APK1-4 (residues 1-438 of Pg) and APK1-5 (residues 1-543 of Pg). Variants of APK1-5 carrying single point mutations in every Lys residue of the AP have also been generated. Analytical ultracentrifugation, differential scanning calorimetry and circular dichroism, have been used to evaluate the hydrodynamic properties, conformational stability and secondary structure alterations of the generated constructs in the presence of Cl- or 6AHA. Our results suggest a critical, but not exclusive, role for kringle 5 and Lys residues 50 and 62 of the AP in the stabilization of the closed conformation of Glu1-Pg. Lys to Ala mutations in residues 50 and 62 of the AP appear to have destabilizing effects in the APK1-5 construct. Direct interaction of AP and isolated kringles was demonstrated and the binding of the AP to Glu1-Pg induces an enhancement in the activation rate of the zymogen. Moreover, the interactions of Pg with PAM (Pg-binding group A streptococcal M-like protein) and its Pg binding motif (VEK30) were characterized. Here, we demonstrate the existence of a partly-open conformational state of Pg when bound to PAM or VEK30. This novel state of Pg can be more effectively activated by Pg activator such as urokinase and streptokinase.
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