| Title: | Identification of residue-to-residue contact between a peptide ligand and its G protein-coupled receptor using periodate-mediated dihydroxyphenylalanine cross-linking and mass spectrometry |
| Author(s): | Umanah GK; Huang L; Ding FX; Arshava B; Farley AR; Link AJ; Naider F; Becker JM; |
| Address: | "Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996, USA" |
| ISSN/ISBN: | 1083-351X (Electronic) 0021-9258 (Print) 0021-9258 (Linking) |
| Abstract: | "Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, alpha-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of alpha-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]alpha-factor, called Bio-DOPA(1)-alpha-factor. This ligand analog was a potent agonist and bound to Ste2p with approximately 65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-alpha-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native alpha-factor or an alpha-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-alpha-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the alpha-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-alpha-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of alpha-factor binding to Ste2p is proposed. The model for bound alpha-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway" |
| Keywords: | "Binding, Competitive Cross-Linking Reagents/chemistry Dihydroxyphenylalanine/*chemistry Kinetics Ligands Mass Spectrometry/methods Mitogens/chemistry Mutagenesis, Site-Directed Peptides/chemistry Periodic Acid/*pharmacology Protein Binding Protein Conform;" |
| Notes: | "MedlineUmanah, George K E Huang, Liyin Ding, Fa-xiang Arshava, Boris Farley, Adam R Link, Andrew J Naider, Fred Becker, Jeffrey M eng R01 GM022087/GM/NIGMS NIH HHS/ R56 GM022087/GM/NIGMS NIH HHS/ GM22087/GM/NIGMS NIH HHS/ GM064779/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural 2010/10/07 J Biol Chem. 2010 Dec 10; 285(50):39425-36. doi: 10.1074/jbc.M110.149500. Epub 2010 Oct 4" |
