TY - JOUR

T1 - Dynamics of urokinase receptor interaction with Peptide antagonists studied by amide hydrogen exchange and mass spectrometry

AU - Jørgensen, Thomas J D

AU - Gårdsvoll, Henrik

AU - Danø, Keld

AU - Roepstorff, Peter

AU - Ploug, Michael

PY - 2004/11/30

Y1 - 2004/11/30

N2 - 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.

AB - 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.

KW - Amides

KW - Animals

KW - CHO Cells

KW - Cricetinae

KW - Cricetulus

KW - Deuterium Oxide

KW - Drosophila

KW - Humans

KW - Hydrogen

KW - Hydrogen Bonding

KW - Kinetics

KW - Ligands

KW - Mass Spectrometry

KW - Models, Chemical

KW - Mutation

KW - Peptides

KW - Protein Structure, Tertiary

KW - Receptors, Cell Surface

KW - Receptors, Urokinase Plasminogen Activator

KW - Recombinant Proteins

KW - Spectrometry, Mass, Electrospray Ionization

KW - Surface Plasmon Resonance

KW - Urokinase-Type Plasminogen Activator

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1021/bi048706j

DO - 10.1021/bi048706j

M3 - Journal article

C2 - 15554712

VL - 43

SP - 15044

EP - 15057

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 47

ER -