Double-Mutant Cycle Scanning of the Interaction of a Peptide Ligand and its G Protein-Coupled Receptor †

Determination of contacts between peptide ligands and their cognate G protein-coupled receptors (GPCRs)2 and the respective energetics of these interactions is essential for understanding how binding is transduced to intracellular signaling. Many studies have employed structure-activity relationships involving ligand analogs and mutagenesis of receptors to discern receptor-ligand interactions (reviewed in refs.1,2). Complementary investigations have used photocrosslinking to provide biochemical evidence for contacts (3, 4). In the 1980s, Fersht and coworkers and Horovitz introduced double-mutant cycle analysis to provide thermodynamic evidence for the interaction between groups within one protein or in ligand-protein complexes (5-7). The method has been applied to a number of receptor systems including the ligand-gated ion channel nicotinic (8, 9) and the GPCR muscarinic (10) acetylcholine receptors. The concept of this method is illustrated in a cycle for a ligand binding to its GPCR (Figure 1A). If the effect on the binding free energy (or the free energy of some other process) of the double mutation is not equal to the sum of effects of the single mutations then the two residues are coupled. Non-zero pairwise coupling energies calculated from such cycles reflect interactions that can be either direct or indirect. Coupling energies found for two directly interacting residues can be converted to a distance constraint and in this respect are similar in nature to nuclear Overhauser connectivities (11). Thus, detailed knowledge of coupling energies for a ligand and a receptor can be used to dock the ligand into a receptor whose structure is available (12-14). Figure 1 Thermodynamic mutant cycles for alanine scanned α-factor analogs interacting with Ste2p. A) Double-mutant cycle to study energetics of the interaction between a given residue X in α-factor with residue Y in the Ste2p receptor. B) Triple-mutant ... We have been studying the biology of a yeast mating factor GPCR, Ste2p, and its contacts with α-factor [Trp1-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr13], the tridecapeptide ligand of this GPCR, by employing α-factor analogs, site-specific mutagenesis and photocrosslinking analysis (15-18). These studies together with extensive molecular biology investigations (19-23) have indicated likely interactions between Y266 and residues near the amine terminus of α-factor. Very recently, biochemical evidence based on disulfide crosslinking indicated that N205 and Y266 might be close in an activated state of Ste2p as represented by a constitutively active mutant of this GPCR (24). In order to probe the thermodynamics of the interactions that occur between α-factor and Ste2p, we have now conducted a double-mutant cycle analysis using binding data previously obtained for the interaction of a series of alanine-scanned α-factor analogs with the N205A and Y266A receptor mutants (16, 24). Noteworthy is the fact that both of these receptor mutants are deficient in signaling but bind α-factor with nM affinity. We also report new studies on the interactions between wild-type and mutant Ste2p with α-factor analogs in which two native residues were replaced by alanine. These double alanine mutants were used to construct triple-mutant thermodynamic cycles (7) to ascertain whether cooperative interactions between residues within α-factor occur during binding to its GPCR. The results show the value of using an alanine-scanned series of a medium sized peptide ligand in multiple-mutant cycle analysis of its interaction with a GPCR and provide thermodynamic values for the interaction free energies of specific residues in α-factor and Ste2p.