title: Structural investigations of recombinant urokinase growth factor-like domain.
abstract: Urokinase-type plasminogen activator (uPA) is a serine protease that converts the plasminogen zymogen into the enzymatically active plasmin. uPA is synthesized and secreted as the single-chain molecule (scuPA) composed of an N-terminal domain (GFD) and kringle (KD) and C-terminal proteolytic (PD) domains. Earlier, the structure of ATF (which consists of GFD and KD) was solved by NMR (A. P. Hansen et al. (1994) Biochemistry, 33, 4847-4864) and by X-ray crystallography alone and in a complex with the soluble form of the urokinase receptor (uPAR, CD87) lacking GPI (C. Barinka et al. (2006) J. Mol. Biol., 363, 482-495). According to these data, GFD contains two β-sheet regions oriented perpendicularly to each other. The area in the GFD responsible for binding to uPAR is localized in the flexible Ω-loop, which consists of seven amino acid residues connecting two strings of antiparallel β-sheet. It was shown by site-directed mutagenesis that shortening of the Ω-loop length by one amino acid residue leads to the inability of GFD to bind to uPAR (V. Magdolen et al. (1996) Eur. J. Biochem., 237, 743-751). Here we show that, in contrast to the above-mentioned studies, we found no sign of the β-sheet regions in GFD in our uPA preparations either free or in a complex with uPAR. The GFD seems to be a rather flexible and unstructured domain, demonstrating in spite of its apparent flexibility highly specific interaction with uPAR both in vitro and in cell culture experiments. Circular dichroism, tryptophan fluorescence during thermal denaturation of the protein, and heteronuclear NMR spectroscopy of ¹⁵N/¹³C-labeled ATF both free and in complex with urokinase receptor were used to judge the secondary structure of GFD of uPA.
authors.affiliation: Russian Cardiology Research and Production Complex, 3-ya Cherepkovskaya ul. 15a, 121552 Moscow, Russia.
authors.forename: I B
authors.initials: IB
authors.lastname: Beloglazova
authors.forename: R Sh
authors.initials: RSh
authors.lastname: Beabealashvilli
authors.forename: Ya G
authors.initials: YG
authors.lastname: Gursky
authors.forename: E V
authors.initials: EV
authors.lastname: Bocharov
authors.forename: K S
authors.initials: KS
authors.lastname: Mineev
authors.forename: E V
authors.initials: EV
authors.lastname: Parfenova
authors.forename: V A
authors.initials: VA
authors.lastname: Tkachuk
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D055293:Receptors, Urokinase Plasminogen Activator / Q000737:chemistry / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D055293:Receptors, Urokinase Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator