Solution Structure of the Amino-Terminal Fragment of Urokinase-Type Plasminogen Activator

Abstract

The amino-terminal fragment (ATF) of urokinase-type plasminogen activator is a two domain protein which consists of a growth factor and a kringle domain. The 1H, 13C, and 15N chemical shifts of this protein have been assigned using heteronuclear two- and three-dimensional NMR experiments on selective and uniformly 15N- and 15N/13C-labeled protein isolated from mammalian cells that overexpress the protein. The chemical shift assignments were used to interpret the NOE data which resulted in a total of 1299 NOE restraints. The NOE restraints were used along with 27 phi angle restraints and 21 hydrogen-bonding restraints to produce 15 low energy structures. The individual domains in the structures are highly converged, but the two domains are structurally independent. The root mean square deviations (rmsd) between residues 11-46 in the growth factor domain and the mean atomic coordinates were 0.99 +/- 0.2 for backbone heavy atoms and 1.65 +/- 0.2 for all non-hydrogen atoms. For residues 55-130 in the kringle domain, the rmsd was 0.84 +/- 0.2 for backbone heavy atoms and 1.42 +/- 0.2 for all non-hydrogen atoms. The overall structures of the individual domains are very similar to the structures of homologous proteins. However, important structural differences between the growth factor and other homologous proteins were observed in the region which has been implicated in binding the urokinase receptor which may explain, in part, why other growth factors show no appreciable affinity for the urokinase receptor.

Mapping Part of the Functional Epitope for Ligand Binding on the Receptor for Urokinase-type Plasminogen Activator by Site-directed Mutagenesis

“…However, in common with other protein-protein interactions studied by alanine scanning mutagenesis (32, 44 -46), only moderate effects were observed on the association rate constants (less than 2.5-fold, see (Table II). A comparable change in the free energy of ligand binding (⌬⌬G) was also observed for the interaction between these suPAR mutants and immobilized prouPA (data not shown), which is in accordance with the location of the epitope for receptor binding on the structurally autonomous, growth factor-like module of uPA (28,(47)(48)(49). Three of the receptor residues critical for uPA binding (Arg 53 , Tyr 57 , and Leu 66 ) have previously been assigned to the structural epitope on uPAR for ligand binding, i.e.…”

Structure-based Engineering of Species Selectivity in the Interaction between Urokinase and Its Receptor

“…and kringle (0.72 Å), which signify a low flexibility of the linker region between these modules in the receptor-bound state as opposed to the flexible nature observed for hATF in solution by NMR (46).…”

Urokinase-Type Plasminogen Activator Potentiates Lipopolysaccharide-Induced Neutrophil Activation

“…3 is a serine protease that catalyzes the conversion of plasminogen to plasmin (1)(2)(3). Human uPA is secreted by many cell populations, including neutrophils and endothelial and epithelial cells, as a single-chain proenzyme (pro-uPA) that possesses little or no proteolytic activity (4 -7).…”