Discovery of a novel conformational equilibrium in urokinase-type plasminogen activator

Sci Rep. 2017 Jun 13;7(1):3385. doi: 10.1038/s41598-017-03457-7.

Abstract

Although trypsin-like serine proteases have flexible surface-exposed loops and are known to adopt higher and lower activity conformations, structural determinants for the different conformations have remained largely obscure. The trypsin-like serine protease, urokinase-type plasminogen activator (uPA), is central in tissue remodeling processes and also strongly implicated in tumor metastasis. We solved five X-ray crystal structures of murine uPA (muPA) in the absence and presence of allosteric molecules and/or substrate-like molecules. The structure of unbound muPA revealed an unsuspected non-chymotrypsin-like protease conformation in which two β-strands in the core of the protease domain undergoes a major antiparallel-to-parallel conformational transition. We next isolated two anti-muPA nanobodies; an active-site binding nanobody and an allosteric nanobody. Crystal structures of the muPA:nanobody complexes and hydrogen-deuterium exchange mass spectrometry revealed molecular insights about molecular factors controlling the antiparallel-to-parallel equilibrium in muPA. Together with muPA activity assays, the data provide valuable insights into regulatory mechanisms and conformational flexibility of uPA and trypsin-like serine proteases in general.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibody Specificity
  • Binding Sites
  • Catalytic Domain
  • Crystallography, X-Ray
  • Mice
  • Models, Molecular
  • Protein Conformation*
  • Single-Domain Antibodies / chemistry*
  • Single-Domain Antibodies / metabolism*
  • Urokinase-Type Plasminogen Activator / chemistry*
  • Urokinase-Type Plasminogen Activator / metabolism*

Substances

  • Single-Domain Antibodies
  • Urokinase-Type Plasminogen Activator