Characterization of the allosteric inhibition of a protein-protein interaction by mass spectrometry.
The allosteric inhibition of the lymphocyte function associated antigen-1/intercellullar adhesion molecule (LFA-1/ICAM-1) interaction, by a class of small molecules, is characterized by a battery of mass spectrometric techniques. Binding of hydantoins to the I domain of LFA-1 is observed by size exclusion chromatography/mass spectrometry (SEC/MS) and by direct electrospray ionization mass spectrometry (ESI/MS). A photoactive hydantoin analog specifically labels an amino acid residue of LFA-1 I domain. Competition with this photoaffinity labeling by a panel of inhibitors is correlated with their Kd's for inhibition of the LFA-1/ICAM interaction. Alterations to the tertiary structure of LFA-1 I domain, upon compound binding, are inferred from perturbation in the ESI mass spectrum of the polypeptide's charge state distribution and by an altered level of nonspecific multimer formation. The results demonstrate specific, stoichiometric, reversible binding of the hydantoins to LFA-1. They further show correlation of this binding with activity and indicate alterations in the polypeptide's tertiary structure, on hydantoin binding, consistent with the proposed mechanism for inhibition of the protein-protein interaction.
Davidson
Walter
W
Research and Development Center, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut 06877-0368, USA. wdavidso@rdg.boehringer-ingelheim.com
Hopkins
Jerry L
JL
Jeanfavre
Deborah D
DD
Barney
Kathleen Last
KL
Kelly
Terence A
TA
Grygon
Christine A
CA
D001667:Binding, Competitive; D006827:Hydantoins / Q000737:chemistry; D007202:Indicators and Reagents; D018799:Intercellular Adhesion Molecule-1 / Q000737:chemistry; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
D006827:Hydantoins; D007202:Indicators and Reagents; D016169:Lymphocyte Function-Associated Antigen-1; D011506:Proteins; D011994:Recombinant Proteins; D018799:Intercellular Adhesion Molecule-1