Ligand binding modulates the structural dynamics and activity of urokinase-type plasminogen activator: A possible mechanism of plasminogen activation

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DOI

  • Tobias Kromann-Hansen, University of California, San Diego
  • ,
  • Eva Louise Lange
  • ,
  • Ida K. Lund, Copenhagen University Hospital, Københavns Universitet
  • ,
  • Gunilla Høyer-Hansen, Copenhagen University Hospital, Københavns Universitet
  • ,
  • Peter A. Andreasen
  • ,
  • Elizabeth A. Komives, University of California, San Diego

The catalytic activity of trypsin-like serine proteases is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. A trypsin-like serine protease of particular interest is urokinasetype plasminogen activator (uPA), which is involved in extracellular tissue remodeling processes. Herein, we used hydrogen/deuterium exchange mass spectrometry (HDXMS) to study regulation of activity in the catalytic domain of the murine version of uPA (muPA) by two muPA specific monoclonal antibodies. Using a truncated muPA variant (muPA16-243), containing the catalytic domain only, we show that the two monoclonal antibodies, despite binding to an overlapping epitope in the 37s and 70s loops of muPA16-243, stabilize distinct muPA16-243 conformations. Whereas the inhibitory antibody, mU1 was found to increase the conformational flexibility of muPA16-243, the stimulatory antibody, mU3, decreased muPA16-243 conformational flexibility. Furthermore, the HDXMS data unveil the existence of a pathway connecting the 70s loop to the active site region. Using alanine scanning mutagenesis, we further identify the 70s loop as an important exosite for the activation of the physiological uPA substrate plasminogen. Thus, the data presented here reveal important information about dynamics in uPA by demonstrating how various ligands can modulate uPA activity by mediating long-range conformational changes. Moreover, the results provide a possible mechanism of plasminogen activation.

OriginalsprogEngelsk
Artikelnummere0192661
TidsskriftP L o S One
Vol/bind13
Nummer2
ISSN1932-6203
DOI
StatusUdgivet - 8 feb. 2018

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