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
| Autor: | Jeffrey M. Becker, Fred Naider, Li-Yin Huang, Adam R. Farley, Andrew J. Link, Boris Arshava, Fa-Xiang Ding, George K. Essien Umanah |
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| Rok vydání: | 2010 |
| Předmět: |
Protein Conformation
Peptide Saccharomyces cerevisiae Plasma protein binding Ligands Tandem mass spectrometry Binding Competitive Biochemistry Mass Spectrometry Receptors G-Protein-Coupled Residue (chemistry) Protein structure Molecular Biology G protein-coupled receptor chemistry.chemical_classification Peptide analog Chemistry Periodic Acid Cell Biology Ligand (biochemistry) Dihydroxyphenylalanine Kinetics Cross-Linking Reagents Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Mutagenesis Site-Directed Mitogens Peptides Protein Binding Signal Transduction |
| Zdroj: | Journal of Biological Chemistry. 285:39425-39436 |
| ISSN: | 0021-9258 |
| Popis: | 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, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of α-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)]α-factor, called Bio-DOPA(1)-α-factor. This ligand analog was a potent agonist and bound to Ste2p with ∼65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-α-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 α-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)-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway. |
| Databáze: | OpenAIRE |
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