The Light Chain Allosterically Enhances the Protease Activity of Murine Urokinase-Type Plasminogen Activator.

ABSTRACT

The active form of the murine urokinase-type plasminogen activator (muPA) is formed by a 27-residue disordered light chain connecting the amino-terminal fragment (ATF) with the serine protease domain. The two chains are tethered by a disulfide bond between C1CT in the disordered light chain and C122CT in the protease domain. Previous work showed that the presence of the disordered light chain affected the inhibition of the protease domain by antibodies. Here we show that the disordered light chain induced a 3.7-fold increase in kcat of the protease domain of muPA. In addition, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and accelerated molecular dynamics (AMD) were performed to identify the interactions between the disordered light chain and the protease domain. HDX-MS revealed that the light chain is contacting the 110s, the turn between the ß10- and ß11-strand, and the ß7-strand. A reduction in deuterium uptake was also observed in the activation loop, the 140s loop and the 220s loop, which forms the S1-specificty pocket where the substrate binds. These loops are further away from where the light chain seems to be interacting with the protease domain. Our results suggest that the light chain most likely increases the activity of muPA by allosterically favoring conformations in which the specificity pocket is formed. We propose a model by which the allostery would be transmitted through the ß-strands of the ß-barrels to the loops on the other side of the protease domain.