Double-mutant cycle scanning of the interaction of a peptide ligand and its G protein-coupled receptor.
The interaction between the yeast G protein-coupled receptor (GPCR), Ste2p, and its alpha-factor tridecapeptide ligand was subjected to double-mutant cycle scanning analysis by which the pairwise interaction 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 only a 2.5-fold and 6-fold decrease, respectively, in the affinity for alpha-factor. The analysis shows that residues at the amine terminus of alpha-factor interact strongly with N205 and Y266 whereas residues in the center and 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 pairwise interactions between residues of the alpha-factor peptide changed upon binding to Ste2p. Strong positive cooperativity between residues 1 through 4 of alpha-factor was observed during receptor binding. In contrast, no thermodynamic evidence was found for an interaction between a residue near the carboxyl terminus of alpha-factor (position 11) and one at the N-terminus (position 3). The study shows that multiple-mutant cycle analyses of the binding of an alanine-scanned peptide to wild-type and mutant GPCRs can provide detailed information on contributions of inter- and intramolecular interactions to the binding energy and potentially prove useful in developing 3D models of ligand docked to its receptor.
Naider
Fred
F
The College of Staten Island and Macromolecular Assemblies Institute of the City University of New York, Staten Island, New York 10314, USA. naider@mail.csi.cuny.edu
Becker
Jeffrey M
JM
Lee
Yong-Hun
YH
Horovitz
Amnon
A
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D008024:Ligands; D000072235:Mating Factor; D010455:Peptides / Q000378:metabolism*; D051964:Receptors, Mating Factor / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D013816:Thermodynamics; D014443:Tyrosine / Q000378:metabolism
D008024:Ligands; D010455:Peptides; D051964:Receptors, Mating Factor; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; D000072235:Mating Factor; D000409:Alanine