Comparative NMR analysis of an 80-residue G protein-coupled receptor fragment in two membrane mimetic environments

Fragments of integral membrane proteins have been used to study the physical chemical properties of regions of transporters and receptors. Ste2p(G31-T110) is an 80-residue polypeptide which contains a portion of the N-terminal domain, transmembrane domain 1 (TM1), intracellular loop 1, TM2 and part of extracellular loop 1 of the α-factor receptor (Ste2p) from Saccharomyces cerevisiae. The structure of this peptide was previously determined to form a helical hairpin in lyso-palmitoylphosphatidyl-glycerol micelles (LPPG) [1]. Herein, we perform a systematic comparison of the structure of this protein fragment in micelles and trifluoroethanol (TFE):water in order to understand whether spectra recorded in organic:aqueous medium can facilitate the structure determination in a micellar environment. Using uniformly labeled peptide and peptide selectively protonated on Ile, Val and Leu methyl groups in a perdeuterated background and a broad set of 3D NMR experiments we assigned 89% of the observable atoms. NOEs and chemical shift analysis were used to define the helical regions of the fragment. Together with constraints from paramagnetic spin labeling, NOEs were used to calculate a transiently folded helical hairpin structure for this peptide in TFE:water. Correlation of chemical shifts was insufficient to transfer assignments from TFE:water to LPPG spectra in the absence of further information.     

Expression and biophysical analysis of two double-transmembrane domain-containing fragments from a yeast G protein-coupled receptor

Fragments of G protein-coupled receptors (GPCRs) are widely used as models to investigate these polytopic integral-membrane, signal-transducing molecules, but have proven difficult to prepare in quantities necessary for NMR analyses. We report on the biosynthesis of two double transmembrane (TM) containing fragments of Ste2p, the alpha-factor GPCR from the yeast Saccharomyces cerevisiae. Ste2p(G31-T110) [TM1-TM2] and Ste2p(R231-S339) [TM6-TM7-CT40] were expressed as TrpDeltaLE fusion proteins in Escherichia coli and released by CNBr cleavage. Expression yields were optimized using different strains and induction parameters, and by performing CNBr cleavage directly on inclusion bodies. Nonlabeled and uniformly labeled [15N]-TM1-TM2 and TM6-TM7-CT40, as well as uniformly labeled [15N,13C]-TM1-TM2 and TM1-TM2 selectively labeled with [15N-Ala], [15N-Phe], [15N-Leu], [15N-Ile], and [15N-Val] were prepared. Yields of target peptides with >95% homogeneity varied from 3 mg/L of fermentation ([15N]-TM6-TM7-CT40) to 20 mg/L (selectively labeled TM1-TM2). The high level biosynthesis and the efficient CNBr processing and purification yields allowed the initiation of a comprehensive biophysical analysis of TM1-TM2 and TM6-TM7-CT40. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that TM1-TM2 was monomeric in this micellar environment, whereas TM6-TM7-CT40 migrated as a dimer. CD analysis indicated that TM1-TM2 was highly helical in SDS and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)], but had a tendency to aggregate in dodecylphosphocholine micelles. Similar results were found with TM6-TM7-CT40. Conditions for NMR measurements were optimized, and both TM1-TM2 and TM6-TM7-CT40 exhibited more than 90% of the expected crosspeaks in the [15N,1H]-HSQC spectrum. These findings set the stage for the determination of the 3D structure of these large domains of a GPCR in micelles using high-resolution NMR.     

Expression and spectroscopic characterization of a large fragment of the μ-opioid receptor

We report here a procedure for the production in Escherichia coli and subsequent purification and characterization of an 80-residue fragment of the human μ-opioid receptor. The fragment (‘TM2–3’), which comprises the second and third transmembrane segments as well as the first extracellular loop of the receptor, was expressed as a fusion with glutathione-S-transferase. The fusion protein, which accumulated in insoluble inclusion bodies, was solubilized with N-lauroylsarcosine, and TM2–3 was obtained by thrombin cleavage of the fusion protein followed by reversed-phase HPLC purification. CD spectroscopy of TM2–3 in lysophosphatidylcholine micelles showed that TM2–3 adopts ～50% α-helical structure in this environment, with the remainder consisting of disordered and/or β-structure. This is consistent with the assumption of an α-helical structure by the two membrane-spanning regions and a nonhelical structure in the loop region of TM2–3. Fluorescence spectroscopy and fluorescence quenching experiments suggested that the extracellular loop lies near the surface of the lysophosphatidylcholine micelle. Our work shows that the study of large receptor fragments is a technically accessible approach to the study of the structural properties of the μ-opioid receptor and, possibly, other G-protein-coupled receptors as well.     

Expression and spectroscopic characterization of a large fragment of the mu-opioid receptor

We report here a procedure for the production in Escherichia coli and subsequent purification and characterization of an 80-residue fragment of the human mu-opioid receptor. The fragment ('TM2-3'), which comprises the second and third transmembrane segments as well as the first extracellular loop of the receptor, was expressed as a fusion with glutathione-S-transferase. The fusion protein, which accumulated in insoluble inclusion bodies, was solubilized with N-lauroylsarcosine, and TM2-3 was obtained by thrombin cleavage of the fusion protein followed by reversed-phase HPLC purification. CD spectroscopy of TM2-3 in lysophosphatidylcholine micelles showed that TM2-3 adopts approximately 50% alpha-helical structure in this environment, with the remainder consisting of disordered and/or beta-structure. This is consistent with the assumption of an alpha-helical structure by the two membrane-spanning regions and a nonhelical structure in the loop region of TM2-3. Fluorescence spectroscopy and fluorescence quenching experiments suggested that the extracellular loop lies near the surface of the lysophosphatidylcholine micelle. Our work shows that the study of large receptor fragments is a technically accessible approach to the study of the structural properties of the mu-opioid receptor and, possibly, other G-protein-coupled receptors as well.     

Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling

Saccharomyces cerevisiae haploid cells communicate with their opposite mating type through peptide pheromones (alpha-factor and a-factor) that activate G protein-coupled receptors (GPCRs). S. cerevisiaewas used as a model system for the study of peptide-responsive GPCRs. Here, we detail the synthesis and characterization of a number of alpha-factor (Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr) pheromone analogues containing the photo-cross-linkable group 4-benzoyl-L-phenylalanine (Bpa). Following characterization, one analogue, [Bpa(1), Tyr(3), Arg(7), Phe(13)]alpha-factor, was radioiodinated and used as a probe for Ste2p, the GPCR for alpha-factor. Binding of the di-iodinated probe was saturable (K(d) = 200 nM) and competable by alpha-factor. Cross-linking into Ste2p was specific for this receptor and reversed by the wild-type pheromone. Chemical and enzymatic cleavage of the receptor/radioprobe complex indicated that cross-linking occurred on a portion of Ste2p spanning residues 251-294 which encompasses transmembrane domain 6, the extracellular loop between transmembrane domains 6 and 7, and transmembrane domain 7. This fragment was verified using T7-epitope-tagged Ste2p and a biotinylated, photoactivatable alpha-factor. After cross-linking with the biotinylated photoprobe and trypsin cleavage, the cross-linked receptor fragment was revealed by both an anti T7-epitope antibody and a biotin probe. This is the first determination of a specific contact region between a Class IV GPCR and its ligand. The results demonstrate that Bpa alpha-factor probes are useful in determining contacts between alpha-factor and Ste2p and initiate mapping of the ligand binding site of this GPCR.     

Biosynthesis and NMR analysis of a 73-residue domain of a Saccharomyces cerevisiae G protein-coupled receptor

The yeast Saccharomyces cerevisiae alpha-factor pheromone receptor (Ste2p) was used as a model G protein-coupled receptor (GPCR). A 73-mer multidomain fragment of Ste2p (residues 267-339) containing the third extracellular loop, the seventh transmembrane domain, and 40 residues of the cytosolic tail (E3-M7-24-T40) was biosynthesized fused to a carrier protein. The multidomain fusion protein (designated M7FP) was purified to near homogeneity as judged by HPLC and characterized by mass spectrometry. In minimal medium, 30-40 mg of M7FP were obtained per liter of culture. The 73-residue peptide was released from its carrier by CNBr and obtained in wild-type, (15)N, and (13)C/(15)N forms. The E3-M7-24-T40 peptide integrated into 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] and dodecylphosphocholine micelles at concentrations (200-500 microM) suitable for NMR investigations. HSQC experiments performed in organic solvents and detergent micelles on (15)N-labeled E3-M7-24-T40 showed a clear dispersion of the nitrogen-amide proton correlation cross-peaks indicative of a pure, uniformly labeled molecule that assumed a partially ordered structure. NOE connectivities, chemical shift indices, J-coupling analysis, and structural modeling suggested that in trifluoroethanol/water (1:1) helical subdomains existed in both the transmembrane and cytoslic tail of the multidomain peptide. Similar conclusions were reached in chloroform/methanol/water (4:4:1). As the cytosolic tail participates in down-regulation of Ste2p, the helical regions in the Ste2p tail may play a role in protein-protein interactions involved in endocytosis.     

Changes in conformation at the cytoplasmic ends of the fifth and sixth transmembrane helices of a yeast G protein-coupled receptor in response to ligand binding

The third intracellular loop (IL3) of G protein-coupled receptors (GPCRs) is an important contact domain between GPCRs and their G proteins. Previously, the IL3 of Ste2p, a Saccharomyces cerevisiae GPCR, was suggested to undergo a conformational change upon activation as detected by differential protease susceptibility in the presence and absence of ligand. In this study using disulfide cross-linking experiments we show that the Ste2p cytoplasmic ends of helix 5 (TM5) and helix 6 (TM6) that flank the amino and carboxyl sides of IL3 undergo conformational changes upon ligand binding, whereas the center of the IL3 loop does not. Single Cys substitution of residues in the middle of IL3 led to receptors that formed high levels of cross-linked Ste2p, whereas Cys substitution at the interface of IL3 and the contiguous cytoplasmic ends of TM5 and TM6 resulted in minimal disulfide-mediated cross-linked receptor. The alternating pattern of residues involved in cross-linking suggested the presence of a 3(10) helix in the middle of IL3. Agonist (WHWLQLKPGQPNleY) induced Ste2p activation reduced cross-linking mediated by Cys substitutions at the cytoplasmic ends of TM5 and TM6 but not by residues in the middle of IL3. Thus, the cytoplasmic ends of TM5 and TM6 undergo conformational change upon ligand binding. An α-factor antagonist (des-Trp, des-His-α-factor) did not influence disulfide-mediated Ste2p cross-linking, suggesting that the interaction of the N-terminus of α-factor with Ste2p is critical for inducing conformational changes at TM5 and TM6. We propose that the changes in conformation revealed for residues at the ends of TM5 and TM6 are affected by the presence of G protein but not G protein activation. This study provides new information about role of specific residues of a GPCR in signal transduction and how peptide ligand binding activates the receptor.     

Structure of a Double Transmembrane Fragment of a G-Protein-Coupled Receptor in Micelles

The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for α-factor of Saccharomyces cerevisiae, was studied in LPPG micelles with the use of solution NMR spectroscopy. The fragment Ste2p(G31-T110) (TM1-TM2) consisted of 19 residues from the N-terminal domain, the first TM helix (TM1), the first cytoplasmic loop, the second TM helix (TM2), and seven residues from the first extracellular loop. Multidimensional NMR experiments on [¹⁵N], [¹⁵N, ¹³C], [¹⁵N, ¹³C, ²H]-labeled TM1-TM2 and on protein fragments selectively labeled at specific amino acid residues or protonated at selected methyl groups resulted in >95% assignment of backbone and side-chain nuclei. The NMR investigation revealed the secondary structure of specific residues of TM1-TM2. TALOS constraints and NOE connectivities were used to calculate a structure for TM1-TM2 that was highlighted by the presence of three α-helices encompassing residues 39-47, 49-72, and 80-103, with higher flexibility around the internal Arg⁵⁸ site of TM1. RMSD values of individually superimposed helical segments 39-47, 49-72, and 80-103 were 0.25 ± 0.10 Å, 0.40 ± 0.13 Å, and 0.57 ± 0.19 Å, respectively. Several long-range interhelical connectivities supported the folding of TM1-TM2 into a tertiary structure typified by a crossed helix that splays apart toward the extracellular regions and contains considerable flexibility in the G⁵⁶VRSG⁶⁰ region. ¹⁵N-relaxation and hydrogen-deuterium exchange data support a stable fold for the TM parts of TM1-TM2, whereas the solvent-exposed segments are more flexible. The NMR structure is consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor.     

Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2(271-326))

Obtaining sufficient amount of purified G-protein coupled receptors (GPCRs) is almost always one of the major challenges for their structural studies. CB2_271–326, a human cannabinoid receptor 2 (CB2) fragment comprising part of the third extracellular loop (EL3), the seventh transmembrane domain (TM7) and C-terminal juxtamembrane region of the receptor, was over-expressed as a fusion protein into inclusion body (IB) of Escherichia coli. The fusion protein was purified by histidine-selected nickel affinity chromatography under denaturing conditions. Then, the fusion protein IBs were solubilized in detergent (Brij58) and the expression fusion leader sequence (TrpLE) was specifically cleaved with tobacco etch virus (TEV) protease. The target fragment, CB2_271–326, was subsequently purified by reverse-phase HPLC and confirmed by SDS–PAGE and mass spectrometry. This hydrophobic fragment can refold in mild detergents digitonin and Brij58. Circular dichroism (CD) spectroscopy of CB2_271–326 in digitonin and Brij58 micelles showed that the fragment adopts a more than 75% α-helical structure, with the remainder having β-strand structure. Fluorescence spectroscopy and quenching studies suggested that the C-terminal region lies near the surface of the digitonin micelles and the TM7 region is folded relatively close to the center of the micelles. This study may provide an alternative strategy for the production and structure/functional studies of GPCRs such as CB2 receptor protein produced in the form of IBs.     

Identification of residue-to-residue contact between a peptide ligand and its G protein-coupled receptor using periodate-mediated dihydroxyphenylalanine cross-linking and mass spectrometry

Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of α-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]α-factor, called Bio-DOPA(1)-α-factor. This ligand analog was a potent agonist and bound to Ste2p with ～65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-α-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the α-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.     

Transverse and tangential orientation of predicted transmembrane fragments 4 and 10 from the human multidrug resistance protein (hMRP1/ABCC1) in membrane mimics

The human multidrug-resistance-associated protein 1 (hMRP1/ABCC1) belongs to the large ATP-binding cassette transporter superfamily. In normal tissues, hMRP1 is involved in tissue defense, whereas, in cancer cells, it is overproduced and contributes to resistance to chemotherapy. We previously investigated the folding properties of the predicted transmembrane fragments (TM) TM16, and TM17 from membrane-spanning domain 2 (MSD2). These TMs folded only partially as an α-helix and were located in the polar headgroup region of detergent micelles used as membrane mimics (Vincent et al. in Biochim Biophys Acta 1768:538–552, 2007; de Foresta et al. in Biochim Biophys Acta 1798:401–414, 2010). We have now extended these studies to TM4 and TM10, from MSD0 and MSD1, respectively. TM10 may be involved in the substrate translocation pathway whereas the role of TM4 is less predictable, because few studies have focused on MSD0, a domain present in some hMRP1 homologs only. Each TM contained a single Trp residue (W142 or W553) acting as an intrinsic fluorescent probe. The location and dynamics of the TMs in dodecylphosphocholine (DPC) or n-dodecyl-β-d-maltoside (DDM) micelles were studied by Trp steady-state and time-resolved fluorescence, including quenching experiments. Overall TM structure was analyzed by far-UV circular dichroism studies in detergent micelles and TFE. TM10 behaved similarly to TM16 and TM17, with an interfacial location in micelles consistent with a possible role in lining the transport pore. By contrast, TM4 behaved like a classical TM fragment with a high α-helical content, and its transmembrane insertion did not require its interaction with other TMs.     

Reconstitution of KCNE1 into lipid bilayers: comparing the structural, dynamic, and activity differences in micelle and vesicle environments

KCNE1 (minK), found in the human heart and cochlea, is a transmembrane protein that modulates the voltage-gated potassium KCNQ1 channel. While KCNE1 has previously been the subject of extensive structural studies in lyso-phospholipid detergent micelles, key observations have yet to be confirmed and refined in lipid bilayers. In this study, a reliable method for reconstituting KCNE1 into lipid bilayer vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (POPG) was developed. Microinjection of the proteoliposomes into Xenopus oocytes expressing the human KCNQ1 (K(V)7.1) voltage-gated potassium channel led to nativelike modulation of the channel. Circular dichroism spectroscopy demonstrated that the percent helicity of KCNE1 is significantly higher for the protein reconstituted in lipid vesicles than for the previously described structure in 1.0% 1-myristoyl-2-hydroxy-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) (LMPG) micelles. SDSL electron paramagnetic resonance spectroscopic techniques were used to probe the local structure and environment of Ser28, Phe54, Phe57, Leu59, and Ser64 of KCNE1 in both POPC/POPG vesicles and LMPG micelles. Spin-labeled KCNE1 cysteine mutants at Phe54, Phe57, Leu59, and Ser64 were found to be located inside POPC/POPG vesicles, whereas Ser28 was found to be located outside the membrane. Ser64 was shown to be water inaccessible in vesicles but found to be water accessible in LMPG micelle solutions. These results suggest that key components of the micelle-derived structure of KCNE1 extend to the structure of this protein in lipid bilayers but also demonstrate the need to refine this structure using data derived from the bilayer-reconstituted protein to more accurately define its native structure. This work establishes the basis for such future studies.     

Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli. Cleavage of a fusion protein with cyanogen bromide.

Both methionine residues in phospholipase A2 (PLA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity. The methionine-less mutant has been expressed as a Cro-LacZ fusion protein in Escherichia coli, from which a pro-PLA2 was liberated by chemical cleavage with CNBr. The general applicability of CNBr cleavage of proteins lacking methionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A2 (HP-PLA2) by a leucine residue, and the introduction of a methionine at a position just preceding the HP-PLA2 sequence. This protein was expressed in E. coli as a 68-kDa Cro-LacZ fusion protein. CNBr cleavage liberated the HP-PLA2 fragment which was reoxidized in vitro. The [Met8----Leu]HP-PLA2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8. p-Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect. The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2-dioctanoyl-sn-glycero-3-phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2-dioctanoyl-sn-glycero-3-phosphoglycol, respectively. In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol greater than phosphatidylethanolamine much greater than phosphatidylcholine. The enzyme has low activity on monomeric 1,2-diheptanoyl-sn-glycero-3-phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration. The binding of the HP-PLA2, porcine pancreatic PLA2 and PLA2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n-hexadecylphosphocholine. The HP-PLA2 has a high apparent Kd (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA2. In mixed micelles of taurodeoxycholate and 1,2-didodecanoyl-sn-glycero-3-phosphocholine, the competitive inhibition of HP-PLA2 by the R and S enantiomers of 2-tetradecanoylaminohexanol-1-phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested. The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors. The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine much less than phosphoethanolamine less than phosphoglycol. The best inhibitor, (R)-2-tetradecanoylaminohexanol-1-phosphoglycol, binds 2200 times stronger than the substrate to the HP-PLA2 active site.     

Structure analysis of the fourth transmembrane domain of Nramp1 in model membranes

Nramp1 (natural resistance-associated macrophage protein 1) is an integral membrane protein with 12 putative transmembrane domains. As a proton-coupled divalent metal cation transporter, it is involved in defense against intracellular pathogens. Disease-causing mutation in Nramp1 occurring at glycine 169 located within the fourth transmembrane domain (TM4) suggests functional importance of this domain. In this paper, we study the three-dimensional structures of a peptide, corresponding to the TM4 of the wild-type Nramp1, in SDS micelles and 2, 2, 2-trifluoroethanol solvent using CD and NMR spectroscopies. We have found that an alpha-helix is predominantly induced in membrane-mimetic environments and the folding of the C-terminal residues is regulated by pH in SDS micelles. The peptide is embedded in SDS micelles and self-associated by coiled-coil interactions. The helix of the peptide in TFE is lengthened towards the N-terminus compared with those in SDS micelles at acidic pH and the self-association of the peptide is also observed in TFE. The fact that Mn(2+) ions are accessible to Asp-14 located in the interior of SDS micelles is found and the binding affinity is increased with increasing pH. The self-association of the peptide may provide a path by which Mn(2+) ions pass through the membrane.     

Structure analysis of the fourth transmembrane domain of Nramp1 in model membranes

Nramp1 (natural resistance-associated macrophage protein 1) is an integral membrane protein with 12 putative transmembrane domains. As a proton-coupled divalent metal cation transporter, it is involved in defense against intracellular pathogens. Disease-causing mutation in Nramp1 occurring at glycine 169 located within the fourth transmembrane domain (TM4) suggests functional importance of this domain. In this paper, we study the three-dimensional structures of a peptide, corresponding to the TM4 of the wild-type Nramp1, in SDS micelles and 2, 2, 2-trifluoroethanol solvent using CD and NMR spectroscopies. We have found that an α-helix is predominantly induced in membrane-mimetic environments and the folding of the C-terminal residues is regulated by pH in SDS micelles. The peptide is embedded in SDS micelles and self-associated by coiled-coil interactions. The helix of the peptide in TFE is lengthened towards the N-terminus compared with those in SDS micelles at acidic pH and the self-association of the peptide is also observed in TFE. The fact that Mn²⁺ ions are accessible to Asp-14 located in the interior of SDS micelles is found and the binding affinity is increased with increasing pH. The self-association of the peptide may provide a path by which Mn²⁺ ions pass through the membrane.     

1H, 15N and 13C backbone resonance assignment of Rv1567c, an integral membrane protein from Mycobacterium tuberculosis

We report here the backbone assignment of Rv1567c, an integral membrane protein from Mycobacterium tuberculosis. The backbone resonance assignments were determined based on triple-resonance experiments with uniformly [¹³C,¹⁵N]-labeled protein in LMPG detergent micelles.     

Identifying functionally important conformational changes in proteins: activation of the yeast α-factor receptor Ste2p

We have developed a procedure in which disulfide cross-links are used to identify regions of proteins that undergo functionally important intramolecular motion. The approach was applied to the identification of disulfide bonds that stabilize the active state of the yeast α-mating pheromone receptor Ste2p, a member of the superfamily of G protein-coupled receptors. Cysteine residues were introduced at random positions in targeted regions of a starting allele of Ste2p that completely lacks cysteines. Libraries of mutated receptors were then screened for alleles that exhibit constitutive signaling. Two strongly activated alleles were recovered containing cysteine residues in transmembrane (TM) segments 5 and 6. Constitutive activity of these alleles was dependent on the presence of both introduced cysteines and was sensitive to reducing agent. Cross-linked peptides derived from the mutant receptors were detected by immunoblotting. Additional sites of cross-linking between TM segments 5 and 6 that did not lead to constitutive activation were also identified. These results indicate that relative motion of the TM segments 5 and 6 in the extracellular half of the membrane is sufficient to activate the receptor and that TM segment 6, but not TM segment 5, exhibits rotational mobility that is not associated with receptor activation.     

In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions.

Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322. The Ste2 and beta la components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in beta la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of beta la; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in beta la secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of beta la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells.     

Yeast Ste2 receptors as tools for study of mammalian protein kinases and adaptors involved in receptor trafficking

Background

Mammalian receptors that couple to effectors via heterotrimeric G proteins (e.g., beta ₂-adrenergic receptors) and receptors with intrinsic tyrosine kinase activity (e.g., insulin and IGF-I receptors) constitute the proximal points of two dominant cell signaling pathways. Receptors coupled to G proteins can be substrates for tyrosine kinases, integrating signals from both pathways. Yeast cells, in contrast, display G protein-coupled receptors (e.g., alpha-factor pheromone receptor Ste2) that have evolved in the absence of receptor tyrosine kinases, such as those found in higher organisms. We sought to understand the motifs in G protein-coupled receptors that act as substrates for receptor tyrosine kinases and the functional consequence of such phosphorylation on receptor biology. We expressed in human HEK 293 cells yeast wild-type Ste2 as well as a Ste2 chimera engineered with cytoplasmic domains of the beta₂-adrenergic receptor and tested receptor sequestration in response to activation of the insulin receptor tyrosine kinase.

Results

The yeast Ste2 was successfully expressed in HEK 293 cells. In response to alpha-factor, Ste2 signals to the mitogen-activated protein kinase pathway and internalizes. Wash out of agonist and addition of antagonist does not lead to Ste2 recycling to the cell membrane. Internalized Ste2 is not significantly degraded. Beta₂-adrenergic receptors display internalization in response to agonist (isoproterenol), but rapidly recycle to the cell membrane following wash out of agonist and addition of antagonist. Beta₂-adrenergic receptors display internalization in response to activation of insulin receptors (i.e., cross-regulation), whereas Ste2 does not. Substitution of the cytoplasmic domains of the β₂-adrenergic receptor for those of Ste2 creates a Ste2/beta₂-adrenergic receptor chimera displaying insulin-stimulated internalization.

Conclusion

Chimera composed of yeast Ste2 into which domains of mammalian G protein-coupled receptors have been substituted, when expressed in animal cells, provide a unique tool for study of the regulation of G protein-coupled receptor trafficking by mammalian receptor tyrosine kinases and adaptor proteins.     

Spectroscopic studies of a phosphoinositide-binding peptide from gelsolin: behavior in solutions of mixed solvent and anionic micelles.

The peptide G(150-169) corresponds to a phosphatidylinositol 4,5-bisphosphate (PIP2) and filamentous actin (F-actin) binding site on gelsolin (residues 150-169, with the sequence KHVVPNEVVVQRLFQVKGRR). The conformation of this peptide in trifluoroethanol (TFE) aqueous solution was determined by 1H nuclear magnetic resonance as the first step toward understanding the structural aspects of the interaction of G(150-169) and PIP2. The circular dichroism experiments show that G(150-169) adopts a predominantly alpha-helical form in both 50% TFE aqueous solution and in the presence of PIP2 micelles, therefore establishing a connection between the two conformations. 1H nuclear magnetic resonance experiments of G(150-169) in TFE co-solvent show that the helical region extends from Pro-154 to Lys-166. The amphiphilic nature of this helical structure may be the key to understanding the binding of the peptide to lipids. Sodium dodecyl sulfate micelle solution is used as a model for anionic lipid environments. Preliminary studies of the conformation of G(150-169) in sodium dodecyl sulfate micelle solution show that the peptide forms an alpha-helix similar to but with some structural differences from that in TFE co-solvent. Fluorescence experiments provide evidence of peptide clustering over a narrow range of peptide/PIP2 ratios, which is potentially relevant to the biological function of PIP2.