Dynamics of urokinase receptor interaction with Peptide antagonists studied by amide hydrogen exchange and mass spectrometry
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Dynamics of urokinase receptor interaction with Peptide antagonists studied by amide hydrogen exchange and mass spectrometry. / Jørgensen, Thomas J D; Gårdsvoll, Henrik; Danø, Keld; Roepstorff, Peter; Ploug, Michael.
In: Biochemistry, Vol. 43, No. 47, 30.11.2004, p. 15044-57.Research output: Contribution to journal › Journal article › Research › peer-review
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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 -
ID: 178219461