The Chain Length Dependence of Helix Formation of the Second Transmembrane Domain of a G Protein-coupled Receptor ofSaccharomyces cerevisiae

Ding, Fa‐Xiang; Schreiber, David; VerBerkmoes, Nathan C.; Becker, Jeffrey M.; Naider, Fred

doi:10.1074/jbc.m111382200

Cited by 19 publications

(19 citation statements)

References 60 publications

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“…Hence, it is tempting to speculate that the side-chains of Gln 5 and Lys 7 face away from the receptor and that this part of the pheromone adopts a β-strand like conformation in its bound state. This proposed orientation of Lys 7 is in agreement with a fluorescence analysis that indicated that the side-chain of Lys 7 most likely faces away from the transmembrane region and interacts with loop residues (28,29).…”

Section: Discussionsupporting

confidence: 86%

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

et al. 2007

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The interaction between the yeast G protein coupled receptor (GPCR), Ste2p, and its α-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 α-factor. The analysis shows that residues at the amine terminus of α-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 α-factor peptide changed upon binding to Ste2p. Strong positive cooperativity between residues 1 through 4 of α-factor was observed during receptor binding. In contrast, no thermodynamic evidence was found for an interaction between a residue near the carboxyl terminus of α-factor (position 11) and one at the N-terminus (position 3). The study shows that multiple-mutant cycle analyses of the binding of an alaninescanned peptide to wild-type and mutant GPCRs can provide detailed information on contributions of inter-and intra-molecular interactions to the binding energy and potentially prove useful in developing 3D models of ligand docked to its 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 † This work was supported by research grants GM22086 and GM22087 from the National Institutes of Health. cycles reflect interactions that can be either direct or indirect. Coupling energies found for two directly interacting residues can be converted to a distance constrain...

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Section: Discussionsupporting

confidence: 86%

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

et al. 2007

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“…Studies in Trifluoroethanol/Water Mixtures-Previous studies on the transmembrane domains of Ste2p were carried out in membrane-mimetic environments including TFE/water, SDS micelles, and various vesicles (13,19,23). In these investigations a fragment corresponding to the seventh transmembrane domain of Ste2p was synthesized as a 30-residue peptide (GT-DVLTTVATLLAVLSLPLSSMWATAANNA) containing 24 residues presumed to represent the hydrophobic core and three residues from the third extracellular loop and the cytosolic tail.…”

Section: Analysis Of Peptidesmentioning

confidence: 99%

“…Recently we showed that the residues at the termini of the second transmembrane domain of Ste2p (a GPCR involved in mating) can affect the integration of this domain into the membrane and can influence the tilt angle of the peptide (19). Specifically, we found that a 30-residue peptide corresponding to transmembrane domain two (TMD-2; 25 residues) plus 5 residues extending into the cytosolic compartment integrated into bilayers was highly helical and tilted at an angle of 34°to the bilayer normal.…”

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confidence: 99%

Synthesis and Biophysical Characterization of a Multidomain Peptide from a Saccharomyces cerevisiae G Protein-coupled Receptor

Naider

Ding

VerBerkmoes

et al. 2003

Journal of Biological Chemistry

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We attached peptides corresponding to the seventh transmembrane domain (TMD7) of the ␣-mating factor receptor (Ste2p) of Saccharomyces cerevisiae to a hydrophilic, 40-residue fragment of the carboxyl terminus of this G protein-coupled receptor. Peptides corresponding to (a) the 40-residue portion of the carboxyl tail (T-40), (b) the tail plus a part of TMD7 (M7-12-T40), and (c) to the tail plus the full TMD7 (M7-24-T40) were chemically synthesized and purified. The molecular mass and primary sequence of these peptides were confirmed by mass spectrometry and tandem mass spectrometry procedures. Circular dichroism (CD) revealed that T-40 was disordered in phosphate buffer and in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-racemic-(1-glycerol)] bilayers. In contrast, M7-12-T40 and M7-24-T40 peptides were partially helical in the presence of vesicles, and difference CD spectroscopy showed that the transmembrane regions of these peptides were 42 and 94% helical, respectively. CD analysis also demonstrated that M7-24-T40 retained its secondary structure in the presence of 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-racemic-(1-glycerol)] micelles at 0.5 mM concentration. Thus, the tail and the transmembrane domain of the multidomain 64-amino acid residue peptide manifest individual conformational preferences. Measurement of tryptophan fluorescence indicated that the transmembrane domain integrated into bilayers in a manner similar to that expected for this region in the native state of the receptor. This study demonstrated that the tail of Ste2p can be used as a hydrophilic template to study transmembrane domain structure using techniques such as CD and NMR spectroscopy.The determination of the structure of G protein-coupled receptors remains an unfulfilled goal of structural biologists with the notable exception of the solution of a high resolution structure of rhodopsin by x-ray crystallography (1). These membrane-bound, information-transducing proteins are acknowledged as a major family of proteins with thousands of representatives encoded by human genes (2, 3). Moreover, the impressive successes in the development of pharmaceuticals targeting G protein-coupled receptors (GPCRs) 1 have created much interest in their structure and mechanism of action (4). Because of the difficulty in obtaining refraction grade crystals of most membrane proteins and their large size, neither x-ray diffraction nor NMR approaches are readily applied to intact receptors. As a route to gain information relevant to these and other membrane proteins such as transporters, many laboratories have taken a reductionist approach and are examining relatively short peptides corresponding to loops and single transmembrane domains (TMDs; Refs. 5-7). A problem encountered in these studies is the poor solubility exhibited by TMDs that leads to difficulty in purifying and conducting biophysical investigations on these molecules. Moreover, although important insights into the conformational preference...

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“…20,21 Given the structural homology of all GPCRs, the commonality of the signal cascade and the vast biochemical and genetic knowledge available on S. cerevisiae, we use the ␣-factor-Ste2p interaction as a paradigm for learning about the interaction of small peptide hormones and their GPCRs. Biophysical studies on the seven transmembrane helices of Ste2p and several loops have been carried out using CD 14,22 and attenuated total reflection Fourier transform infrared spectroscopies 23,24 in organic, organic/aqueous, detergent, and lipid environments. The CD analysis showed that peptides corresponding to the seven transmembrane domains of Ste2p were all helical in trifluoroethanol/water (4:1) but that the percent helicity varied from 85 to 43%.…”

Section: Introductionmentioning

confidence: 99%

High resolution NMR analysis of the seven transmembrane domains of a heptahelical receptor in organic‐aqueous medium

et al. 2002

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The NMR properties of seven peptides representing the transmembrane domains of the alpha-factor receptor from Saccharomyces cerevisiae were examined in trifluoroethanol/water (4:1) at 10 to 55 degrees C. The parameters extracted indicated all peptides were helical in this membrane mimetic solvent. Using chemical shift indices as the criterion, helicity varied from 64 to 83%. The helical residues in the peptides corresponded to the region predicted to cross the hydrocarbon interior of the bilayer. A study of a truncated 25-residue peptide corresponding to domain 2 gave evidence that the helix extended all the way to the N-terminus of this peptide, indicating that sequence and not chain end effects are very important in helix termination for our model peptides. Both nuclear Overhauser effect spectroscopy (NOESY) connectivities and chemical shift indices revealed significant perturbations around prolyl residues in the helices formed by transmembrane domains 6 and 7. Molecular models of the transmembrane domains indicate that helices for domains 6 and 7 are severely kinked at these prolyl residues. The helix perturbation around proline 258 in transmembrane domain 6 correlates with mutations that cause phenotypic changes in this receptor.

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The Chain Length Dependence of Helix Formation of the Second Transmembrane Domain of a G Protein-coupled Receptor ofSaccharomyces cerevisiae

Cited by 19 publications

References 60 publications

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

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

Synthesis and Biophysical Characterization of a Multidomain Peptide from a Saccharomyces cerevisiae G Protein-coupled Receptor

High resolution NMR analysis of the seven transmembrane domains of a heptahelical receptor in organic‐aqueous medium

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