Double-Mutant Cycle Scanning of the Interaction of a Peptide Ligand and Its G Protein-Coupled Receptor
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- 作者：Fred Naider ; Jeffrey M. Becker ; Yong-Hun Lee ; Amnon Horovitz
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：March 20, 2007
- 年：2007
- 卷：46
- 期：11
- 页码：3476 - 3481
- 全文大小：91K
- 年卷期：v.46,no.11(March 20, 2007)
- ISSN：1520-4995
文摘
The interaction between the yeast G protein-coupled receptor (GPCR), Ste2p, and its 
-factortridecapeptide ligand was subjected to double-mutant cycle scanning analysis by which the pairwiseinteraction energy of each ligand residue with two receptor residues, N205 and Y266, was determined.The mutations N205A and Y266A were previously shown to result in deficient signaling but cause onlya 2.5-fold and 6-fold decrease, respectively, in the affinity for -factor. The analysis shows that residuesat the amine terminus of -factor interact strongly with N205 and Y266 whereas residues in the centerand at the carboxyl terminus of the peptide interact only weakly if at all with these receptor residues.Multiple-mutant thermodynamic cycle analysis was used to assess whether the energies of selected pairwiseinteractions between residues of the -factor peptide changed upon binding to Ste2p. Strong positivecooperativity between residues 1 through 4 of -factor was observed during receptor binding. In contrast,no thermodynamic evidence was found for an interaction between a residue near the carboxyl terminusof -factor (position 11) and one at the N-terminus (position 3). The study shows that multiple-mutantcycle analyses of the binding of an alanine-scanned peptide to wild-type and mutant GPCRs can providedetailed information on contributions of inter- and intramolecular interactions to the binding energy andpotentially prove useful in developing 3D models of ligand docked to its receptor.
-factortridecapeptide ligand was subjected to double-mutant cycle scanning analysis by which the pairwiseinteraction energy of each ligand residue with two receptor residues, N205 and Y266, was determined.The mutations N205A and Y266A were previously shown to result in deficient signaling but cause onlya 2.5-fold and 6-fold decrease, respectively, in the affinity for -factor. The analysis shows that residuesat the amine terminus of -factor interact strongly with N205 and Y266 whereas residues in the centerand at the carboxyl terminus of the peptide interact only weakly if at all with these receptor residues.Multiple-mutant thermodynamic cycle analysis was used to assess whether the energies of selected pairwiseinteractions between residues of the -factor peptide changed upon binding to Ste2p. Strong positivecooperativity between residues 1 through 4 of -factor was observed during receptor binding. In contrast,no thermodynamic evidence was found for an interaction between a residue near the carboxyl terminusof -factor (position 11) and one at the N-terminus (position 3). The study shows that multiple-mutantcycle analyses of the binding of an alanine-scanned peptide to wild-type and mutant GPCRs can providedetailed information on contributions of inter- and intramolecular interactions to the binding energy andpotentially prove useful in developing 3D models of ligand docked to its receptor.