FIGURE 6.
Defining the site 2 epitope bin in uPAR DI. Data shown for mAb mR1, recognizing the second dominating immunogenic epitope (site 2), are shown in A. This panel is organized as in Fig. 5 except that the molecular model for ATF-uPAR-SMB is rotated 180° horizontally as illustrated. The hotspot for mR1 binding is shown in blue, and that for the DIII-reactive mAb R2 is shown in cyan for comparison. The sensorgrams in panel B show that uPAR bound to immobilized mR1 does indeed bind the uPA derivatives ATF (red curve) and GFD (blue curve) when these are injected at 200 nm. The formed ternary complexes are, however, relatively more unstable than the binary uPAR-mR1 complex (black curve), leading to a roughly 15-fold increase in the apparent koff for ATF-uPAR-mR1, 25-fold for GFD-uPAR-mR1, and 3-fold for AE234-uPAR-mR1 (data not shown) during injection of the respective ligands at saturating conditions (B). The sensorgrams in panel C show that SMB does bind uPAR-mR1complexes, but this occurs with a moderately decreased affinity (KD is 4.4 ± 0.5 μm) as compared to that measured for uPAR-R2 complexes measured in parallel (KD is 1.8 ± 0.2 μm), as derived from the equilibrium binding isotherms shown in the inset.