Congress of the International Society on Thrombosis and Haemostasis

Crystal structures of a soluble urokinase receptor mutant in its ligand-free form and in complex with its ligand uPA

Abstract number: AS-WE-047

Xu¹ X., Gårdsvoll² H., Ploug² M., Huang¹ M.

¹¹Division of Haemostasis and Thrombosis, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA ²²Finsen Laboratory, Rigshospitalet, Copenhagen Biocenter, Copenhagen, Denmark

How-to-cite Xu X, Gårdsvoll H, Ploug M, Huang M. Crystal structures of a soluble urokinase receptor mutant in its ligand-free form and in complex with its ligand uPA. Journal of Thrombosis and Haemostasis 2009; Volume 7, Supplement 2: Abstract AS-WE-047

The urokinase receptor, uPAR, is a multi-function protein capable of not only focalizing uPA-mediated fibrinolysis to the pericellular microenvironment, but also of promoting cell migration and chemotaxis. Consistent with this multi-functional role, uPAR is reported to bind several extracellular ligands, including uPA, somatomedin B domain of vitronectin, a number of integrins, and GPCR. In recent years, crystal structures of uPAR in complex with the amino-terminal domain (ATF) of uPA, SMB domain of vitronectin and an inhibitory peptide were determined. These structural studies suggested that uPAR possesses some structural flexibility. It is, however, not clear whether this flexibility is an inherent property of the uPAR structure per se or whether it is induced upon ligand binding. Although the crystal structure of human uPAR in its ligand-free state would clarify this issue it remains unfortunately elusive. As a surrogate, we now report the crystal structures of a stabilized human uPAR mutant (H47C/N259C) in its ligand-free form to 2.3 Å and in complex with ATF to 3.2 Å. These crystal structures confirmed the presence of the engineered disulfide bond between residues 47 and 259. The overall structure of this uPAR mutant is quite similar to uPAR in its uPA-binding conformation (RMSD of 2 Å), but different from its peptidyl inhibitor-binding conformation. Despite the overall similarity to uPAR in its ATF binding conformation, there are some significant differences. For example, the loops containing residue 47 and 259 were observed to be pulled closer to each other by the formation of this disulfide. Biochemical studies demonstrate that this suPAR mutant binds ATF with an affinity indistinguishable to that of wild type suPAR showing that this stabilization does not affect the functionality of the ligand binding cavity. Taken together, these studies reveal the plasticity of uPAR itself as well as of uPAR with an occupied ligand binding cavity.

Disclosure of interest: none declared.

To cite this abstract use the following format:

Journal of Thrombosis and Haemostasis 2007; Volume 5, Supplement 2: abstract number

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Session name:	ISTH2009
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