Dynamics of urokinase receptor interaction with Peptide antagonists studied by amide hydrogen exchange and mass spectrometry.
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 activator receptor (uPAR). These experiments reveal that at least six out of eight amide hydrogens in a synthetic nine-mer peptide antagonist (AE105) become sequestered upon engagement in uPAR binding. Various uPAR mutants with decreased affinity for this peptide antagonist gave similar results, thereby indicating that deletion of the favorable interactions involving the side chains of these residues in uPAR does not affect the number of hydrogen bonds established by the main chain of the peptide ligand. The isolated growth factor-like domain (GFD) of the cognate serine protease ligand for uPAR showed 11 protected amide hydrogens in the receptor complex. Interestingly, a naturally occurring O-linked fucose on Thr(18) confers 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 all amide hydrogens on the peptide ligand. This yields bimodal isotope patterns from which dissociation rate constants can be determined. In addition, the distinct bimodal isotope distributions also allow investigation of the exchange kinetics of receptor-bound peptides providing information about the local structural motions at the interface. These exchange experiments therefore provide both structural and kinetic information on the interaction between uPAR and these small peptide antagonists, which in model systems show promise as inhibitors of intravasation of human cancer cells.
Jørgensen
Thomas J D
TJ
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. tjdj@bmb.sdu.dk
Gårdsvoll
Henrik
H
Danø
Keld
K
Roepstorff
Peter
P
Ploug
Michael
M
D000577:Amides / Q000737:chemistry*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D017666:Deuterium Oxide / Q000737:chemistry; D004330:Drosophila / Q000166:cytology; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D006860:Hydrogen Bonding; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D008956:Models, Chemical; D009154:Mutation; D010455:Peptides / Q000037:antagonists & inhibitors*; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D020349:Surface Plasmon Resonance; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism
D000577:Amides; D008024:Ligands; C524898:PLAUR protein, human; D010455:Peptides; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011994:Recombinant Proteins; D006859:Hydrogen; D014568:Urokinase-Type Plasminogen Activator; D017666:Deuterium Oxide