title: Activation of the zymogen to urokinase-type plasminogen activator is associated with increased interdomain flexibility.
abstract: A key regulatory step for serine proteases of the trypsin clan is activation of the initially secreted zymogens, leading to an increase in activity by orders of magnitude. Zymogen activation occurs by cleavage of a single peptide bond near the N-terminus of the catalytic domain. Besides the catalytic domain, most serine proteases have N-terminal A-chains with independently folded domains. Little is known about how zymogen activation affects the interplay between domains. This question is investigated with urokinase-type plasminogen activator (uPA), which has an epidermal growth factor domain and a kringle domain, connected to the catalytic domain by a 15-residue linker. uPA has been implicated under several pathological conditions, and one possibility for pharmacological control is targeting the conversion of the zymogen pro-uPA to active uPA. Therefore, a small-angle X-ray scattering study of the conformations of pro-uPA and uPA in solution was performed. Structural models for the proteins were derived using available atomic-resolution structures for the various domains. Active uPA was found to be flexible with a random conformation of the amino-terminal fragment domain with respect to the serine protease domain. In contrast, pro-uPA was observed to be rigid, with the amino-terminal fragment domain in a fixed position with respect to the serine protease domain. Analytical ultracentrifugation analysis supported the observed difference between pro-uPA and uPA in overall shape and size seen with small-angle X-ray scattering. Upon association of either of two monoclonal Fab (fragment antigen-binding) fragments that are directed against the catalytic domain of, respectively, pro-uPA and uPA, rigid structures were formed.
authors.affiliation: Department of Chemistry and iNANO Interdisciplinary Nanoscience Center, Aarhus University, Langelandsgade 140, DK-8000 Aarhus C, Denmark.
authors.forename: Manja A
authors.initials: MA
authors.lastname: Behrens
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Botkjaer
authors.forename: Sumit
authors.initials: S
authors.lastname: Goswami
authors.forename: Cristiano L P
authors.initials: CL
authors.lastname: Oliveira
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
authors.forename: Christine R
authors.initials: CR
authors.lastname: Schar
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Declerck
authors.forename: Cynthia B
authors.initials: CB
authors.lastname: Peterson
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
authors.forename: Jan Skov
authors.initials: JS
authors.lastname: Pedersen
mesh_terms: D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D057057:Serine Proteases / Q000378:metabolism*; D014461:Ultracentrifugation; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004792:Enzyme Precursors; D057057:Serine Proteases; D014568:Urokinase-Type Plasminogen Activator