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| - Dynamics of Urokinase Receptor Interaction with Peptide Antagonists Studied byAmide Hydrogen Exchange and Mass Spectrometry
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| - Using amide hydrogen exchange combined with electrospray ionization mass spectrometry,we have in this study determined the number of amide hydrogens on several peptides that become solvent-inaccessible as a result of their high-affinity interaction with the urokinase-type plasminogen activatorreceptor (uPAR). These experiments reveal that at least six out of eight amide hydrogens in a syntheticnine-mer peptide antagonist (AE105) become sequestered upon engagement in uPAR binding. VariousuPAR mutants with decreased affinity for this peptide antagonist gave similar results, thereby indicatingthat deletion of the favorable interactions involving the side chains of these residues in uPAR does notaffect the number of hydrogen bonds established by the main chain of the peptide ligand. The isolatedgrowth factor-like domain (GFD) of the cognate serine protease ligand for uPAR showed 11 protectedamide hydrogens in the receptor complex. Interestingly, a naturally occurring O-linked fucose on Thr18confers protection of two additional amide hydrogens in GFD when it forms a complex with uPAR.Dissociation of the uPAR−peptide complexes is accompanied by a correlated exchange of nearly allamide hydrogens on the peptide ligand. This yields bimodal isotope patterns from which dissociation rateconstants can be determined. In addition, the distinct bimodal isotope distributions also allow investigationof the exchange kinetics of receptor-bound peptides providing information about the local structural motionsat the interface. These exchange experiments therefore provide both structural and kinetic information onthe interaction between uPAR and these small peptide antagonists, which in model systems show promiseas inhibitors of intravasation of human cancer cells.
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