Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide

Abstract

We report the crystal structure of a soluble form of human urokinase-type plasminogen activator receptor (uPAR/CD87), which is expressed at the invasive areas of the tumor-stromal microenvironment in many human cancers. The structure was solved at 2.7 A in association with a competitive peptide inhibitor of the urokinase-type plasminogen activator (uPA)-uPAR interaction. uPAR is composed of three consecutive three-finger domains organized in an almost circular manner, which generates both a deep internal cavity where the peptide binds in a helical conformation, and a large external surface. This knowledge combined with the discovery of a convergent binding motif shared by the antagonist peptide and uPA allowed us to build a model of the human uPA-uPAR complex. This model reveals that the receptor-binding module of uPA engages the uPAR central cavity, thus leaving the external receptor surface accessible for other protein interactions (vitronectin and integrins). By this unique structural assembly, uPAR can orchestrate the fine interplay with the partners that are required to guide uPA-focalized proteolysis on the cell surface and control cell adhesion and migration.

Figure 1

The structure of human uPAR. (A)The overall modular structure of uPAR as a ribbon diagram. The individual uPAR domains are assembled in a right-handed orientation and are coloured yellow (DI), blue (DII) and red (DIII). The inset illustrates the typical β-strand nomenclature (Low et al, 1976) for a three-fingered fold, using bucandin as a model (Torres et al, 2001). Adopting this nomenclature, the 17 β-strands of uPAR encompass the regions 2–6 (βIA), 12–16 (βIB), 23–33 (βIC), 36–46 (βID), 53–57 (βIE) and 64–71 (βIF) in DI; 94–96 (βIIA), 112–114 (βIIB), 121–129 (βIIC), 142–149 (βIID), 155–160 (βIIE) and 164–171 (βIIF) in DII; and 189–199 (βIIIA), 211- 216 (βIIIB), 221–229 (βIIIC), 234–242 (βIIID) and 262–266 (βIIIF) in DIII. The short helical stretches in DIII (αIIIE) encompass residues 244–248 and 250–255. The bended β-strands in DII and DIII are indicated separately. (B) From left to right, superimpositions of bucandin (yellow) on uPAR DI and DII (grey) and of CD59 (orange) on uPAR DIII (grey). Disulphide bonds are coloured green in the superimpositions. For sake of clarity, the positions of the N- and C-termini are indicated in panel A.

Figure 2

Molecular surface representation of the uPAR–peptide complex. The surface of uPAR is shown in grey, whereas the bound antagonist peptide (AE147) is shown as a ribbon diagram in dark blue. Receptor residues interacting with AE147 are coloured orange (hydrophobic) or cyan (polar). (A) The front side, (B) the upper side and (C) the rear side of the complex, with asparagine-linked glycosylation sites coloured light green. The C-terminal end is shown in panels A and C. In panel B, it is located behind the molecule and hence it cannot be seen by the reader.

Figure 3

Interacting residues in both the antagonist peptide AE147 and uPAR as observed in the crystal structure of the uPAR–peptide complex. The amino-acid sequence of AE147 is shown in the single-letter code with capitals denoting L-amino acids and lower case D-amino acids. Cha: L-β-cyclohexyl-alanine. Residues in black are from human uPAR (Roldan et al, 1990). The corresponding residues from murine uPAR (see text) are coloured blue (Kristensen et al, 1991). Bold letters in blue highlight nonconservative substitutions among these residues in the cavity of human and murine uPAR.

Figure 4

Stereo view of two 2F_o−F_c omit maps. (A) A 1.5σ level contoured map for the carbohydrate linked to N162 in uPAR domain DII. (B) A similar contoured map for the bound AE147 (in grey). The refined atomic model of AE147 is shown in stick representation (red). Cha: L-β-cyclohexyl-alanine; NAG: N-acetylglucosamine; Man: mannose.

Figure 5

Binding of the antagonist peptide in uPAR cavity. (A) The interface between uPAR (illustrated as surface representation) and AE147 (illustrated by a combined ribbon and stick representation in dark blue) is shown from the front side. The interface provided by uPAR is coloured pale orange (hydrophobic) or cyan (polar), whereas the corresponding side chains in AE147 are coloured red. Note that the indole side chain of W10 efficiently engages the deep and prominent hydrophobic hole of the central cavity of uPAR and the side chain of L9 is located proximate to its entrance. (B) Superposition of all eight peptide molecules observed in the unit cell. The figure shows superposition of their Cα backbones and the side chains of Cha4, F5, s6, Y8, L9 and W10 which are involved in binding to uPAR (see text).

Figure 6

Model of the uPA–uPAR complex. (A) Stereo views of the key residues by which AE147 (blue) and the β-hairpin in GFD of uPA (red) bind to uPAR in our crystal structure and in a GFD–uPAR model, respectively. Note that W10 and L9 of AE147 exhibit a spatial superimposition with Y24 and F25 in uPA. (B) Model for the structure of the human GFD–uPAR complex. The uPAR molecule is shown as a molecular surface representation with the residues interacting with GFD in the same colour code as those used in Figures 2 and 4. The receptor-binding module of uPA is shown in red as a ribbon diagram with the side chains of Y24, F25, I28 and W30, which are highlighted by sticks. A schematic representation of the glycolipid (GPI) anchor is shown to connect the C-terminus of uPAR to a model of the cell membrane. Note that the GPI anchor is not shown to scale and the relative orientation of uPAR to the cell membrane is arbitrarily presented.