Hdxms Reveals the Role of Dynamics on the Serine Protease Activity of the Urokinase-Type Plasminogen Activator (UPA)

Abstract

The urokinase-type plasminogen activator (uPA) is a key regulator of cellular processes such wound healing, angiogenesis and anticoagulation, among others. This protein is a serine protease synthesized as inactive single-chain zymogen that becomes active after a very specific cleavage between K158 and I159. The uPA catalyzes the rate limiting step of anticoagulation: the activation of peri-cellular plasminogen into the active serine protease plasmin. Even though the chemistry behind this reaction is well understood with a Ser/Asp/His catalytic triad, the dynamics of the protein and how dynamics control the activity remain unknown. The uPA is formed by an EGF domain, a Kringle domain, a 27-residue linker region and a serine protease domain. The structure of Full-length uPA has not been solved, however, there are structures available for the EGF and Kringle domains (the Amino terminal fragment, or ATF), and the serine protease domain separately. Whether the ATF interacts through the linker with the protease domain remains unknown, but the full-length protein is 6-fold more active (kcat) towards plasminogen when the ATF is present. We hypothesized that there is through-linker dynamic allostery within uPA. To test our hypothesis, we evaluated the changes in dynamics of uPA by Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS). We compared the protease domain alone to full-length uPA, the single chain to the two chain, and receptor (uPAR)-bound to free. The HDX-MS results help to resolve which regions of the protease domain are allosterically regulated.