Lysine-dependant glu-plasminogen binding to a-enolase induces conformational changes resulting in higher affinity interactions

Human glu-plasminogen (Glu-Plg) is a single chain trypsin-like serine endopeptidase zymogen, composed of both a heavy chain (65 kpa), containing an N-tenninal acidic domain with basic hinge region (proactivation domain), five triple-disulfide-bonded kringle structures on which the lysine binding sites (kringles 1,4 and 5) are located, and a light chain (25 kDa) containing the protease domain. Glu-Plg exhibits a closed conformation with a radius of gyration of 39 A. The acidic pro-activation domain assists in maintaining the closed conformation of the molecule. Removal of this domain results in the formation of lys-Plg which has a more open conformation. Pig interacts with proteins such as a-enolase in a lysine-dependent manner. This is mediated by the lysine binding sites on the kringles of Pig which contain a hydrophobic pocket. This study describes the binding of Glu- and Lys-Plg to immobilised dimeric a-enolase using BlAcore analysis. Binding of Lys-Plg to a-enolase was saturable at 4 mM with an apparent KDl=3-3xlO"M and Krl-lxlO" M. The binding of Glu-Plg to a-enolase was not saturable at 4 mM and had dissociation constants of KDi=4.0xlQ-6M and KD2=8.8xlQ-8M. Titration of Glu-Plg with e-amino caproic acid and a-enolase in the presence of the hydrophobic flurosecence probe 8-anilino-l-napthalene sulfonic acid (ANS) resulted in an initial lysine-dependent ANS flurosecence decrease followed by an increase in ANS fluroscence, as well as a concomittant lysine-dependent increase in its intrinsic fluroscence which was comparable to that of Lys-Plg. Our data suggest that the binding of Glu-Plg to a-enolase may be a multi-step event that involves lysine-mediated conformational changes in Glu-Plg resulting in higher affinity interaction(s) between Glu-Plg and a-enolase.