Cross-Linking of a DOPA-Containing Peptide Ligand into Its G Protein-Coupled Receptor

• Published in: Biochemistry (Easton) 2009-03, Vol.48 (9), p.2033-2044
• Main Authors: Umanah, George K. E, Son, Cagdas, Ding, FaXiang, Naider, Fred, Becker, Jeffrey M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Arginine - chemistry; Arginine - genetics; Arginine - metabolism; Binding Sites - genetics; Cell Membrane - chemistry; Cell Membrane - metabolism; Cross-Linking Reagents - chemistry; Cysteine - chemistry; Cysteine - genetics; Cysteine - metabolism; Dihydroxyphenylalanine - chemistry; Immunoblotting; Ligands; Models, Molecular; Oligopeptides - chemistry; Protein Binding; Protein Structure, Tertiary; Receptors, G-Protein-Coupled - chemistry; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Receptors, Mating Factor - chemistry; Receptors, Mating Factor - genetics; Receptors, Mating Factor - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transformation, Genetic; Tyrosine - chemistry; Tyrosine - genetics; Tyrosine - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi802061z

The interaction between a 3,4-dihydroxyphenylalanine (DOPA) labeled analogue of the tridecapeptide α-factor (W-H-W-L-Q-L-K-P-G-Q-P-M-Y) and Ste2p, a Saccharomyces cerevisiae model G protein-coupled receptor (GPCR), has been analyzed by periodate-mediated cross-linking. Chemically synthesized α-factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine7([Lys7(BioACA),Nle12,DOPA13]α-factor; Bio-DOPA-α-factor) was used for cross-linking into Ste2p. The biological activity of Bio-DOPA-α-factor was about one-third that of native α-factor as determined by growth arrest assay and exhibited about a 10-fold lower binding affinity to Ste2p. Bio-DOPA-α-factor cross-linked into Ste2p as demonstrated by Western blot analysis using a neutravidin-HRP conjugate to detect Bio-DOPA-α-factor. Cross-linking was inhibited by excess native α-factor and an α-factor antagonist. The Ste2p−ligand complex was purified using a metal ion affinity column, and after cyanogen bromide treatment, avidin affinity purification was used to capture Bio-DOPA-α-factor-Ste2p cross-linked peptides. MALDI-TOF spectrometric analyses of the cross-linked fragments showed that Bio-DOPA-α-factor reacted with the Phe55−Met69 region of Ste2p. Cross-linking of Bio-DOPA-α-factor was reduced by 80% using a cysteine-less Ste2p (Cys59Ser). These results suggest an interaction between position 13 of α-factor and residue Cys59 of Ste2p. This study is the first to report DOPA cross-linking of a peptide hormone to a GPCR and the first to identify a residue-to-residue cross-link between Ste2p and α-factor, thereby defining a specific contact point between the bound ligand and its receptor.