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

Tobias Kromann-Hansen,Gunilla Høyer-Hansen,Ida K Lund,Eva Louise Lange,Elizabeth A Komives,Peter A Andreasen,Eugene A Permyakov

doi:10.1371/journal.pone.0192661

Abstract

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 urokinase-type 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.

Highlights

IntroductionUrokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a pivotal role in fibrinolysis in the extracellular space as initiator of a protein cascade eventually leading to generation of another trypsin-serine protease, plasmin
Urokinase-type plasminogen activator is a trypsin-like serine protease that plays a pivotal role in fibrinolysis in the extracellular space as initiator of a protein cascade eventually leading to generation of another trypsin-serine protease, plasmin
As the hydrogen/deuterium exchange mass spectrometry (HDXMS) results reveals a functional link between the 70s loop and the active site region, we investigated if the 70s loop serves as an exosite during cleavage of the physiological Urokinase-type plasminogen activator (uPA) substrate plasminogen

Summary

Introduction

Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a pivotal role in fibrinolysis in the extracellular space as initiator of a protein cascade eventually leading to generation of another trypsin-serine protease, plasmin. Plasmin cleavage of single-chain uPA liberates a new N-terminus (Ile16) that becomes inserted into a hydrophobic binding cleft referred to as the activation pocket. This results in ordering of several surfaceexposed loops, including the N-terminal activation loop (residues 16–21), the 140s loop (residues 142–152), the 180s loop (residues 184–194) and the 220s loop (residues 216–223) [8]. As a result of ordering these loops, the active site of uPA (His, Asp102 and Ser195) including the primary S1 substrate-binding pocket (Asp189) and the oxyanion hole defined by the main chain amides of Gly193 and Ser195, adopts a fully-catalytically active conformation [9]

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Conclusion