Crystal Structure of the Interaction of Human Urokinase Plasminogen Activitor with Its Receptor.

Abstract

Listen

Translate article

Urokinase-type plasminogen activator (uPA) and its cellular receptor (uPAR) mediate plasminogen activation. The uPA binds to uPAR with high affinity (Kd 0.1–1nM), thus localizing the generation of plasmin from plasminogen on the surface of a variety of cells. uPA-uPAR binding is also involved in other cellular functions and diverse pathophysiological processes such as tissue remodeling during wound healing, atherosclerosis, angiogenesis and tumor metastasis. We have determined the structure of uPAR complexed with the amino terminal fragment (amino acid residues 1–143) of uPA (ATF), which contains the uPAR binding domain, at 1.9 Å resolution by X-ray crystallography. Soluble uPAR (suPAR) and the ATF were expressed separately in stably transfected Drosophila Schneider 2 (S2) cells. The suPAR-ATF complex was crystallized by the sitting-drop vapor diffusion method. However, the diffraction of this crystal was limited to 3.1 Å resolution. Therefore, the Fab fragment of an antibody raised against suPAR, ATN-615, was used to facilitate suPAR-ATF crystallization. Crystals of the suPAR-ATF-ATN615 ternary complex were generated by microdialysis with 4% PEG4K, 5% ethylene glycol, 5% methanol, 0.05% sodium azide, 50 mM cacodylate pH 6.5. A complete data set of the ternary complex to 1.9 Å was collected using synchrotron radiation at the Advanced Photon Source (APS), Argonne National Laboratory. The crystals belong to the monoclinic space group, with unit cell parameters a=51.79 Å, b=86.81 Å, c=124.69 Å and β=94.54°. uPAR is comprised of three consecutive domains (D1, D2 and D3) that form the shape of a thick-walled teacup with a diameter of about 52 Å and a height of 27 Å. At the center of teacup and surrounded by three suPAR domains is a cone shape cavity with a wide opening (25 Å) and large depth (14 Å). ATF consists of a growth factor domain (GFD) and a kringle domain. Both domains pack more tightly in the complex structure compared with their unbound state. The GFD domain of uPA occupies part of the uPAR cavity and is primarily responsible for uPAR binding. The D1 domain of uPAR forms three hydrogen bonds and many hydrophobic interactions with the GFD domain of uPA, thus playing an important role in the binding of uPA. However, D2 and D3 of uPAR also have direct interactions with the GFD domain of uPA. The kringle domain of uPA sits outside the uPAR pocket, but forms some direct contacts with the D1 domain of uPAR. Therefore, the three domains of uPAR and two domains of uPA form a complementary interaction, which describes the structural basis for the high affinity binding of uPA to uPAR. This structure presents the first high resolution view of uPA-uPAR interaction, and may provide a new platform to design de novo uPA-uPAR antagonists.