Fragments of the second transmembrane helix of three G-protein-coupled receptors: comparative synthetic, structural and conformational studies (original) (raw)
Related papers
Journal of Peptide Science, 2006
Transmembrane domains (TMDs) of G-protein coupled receptors (GPCRs) have very low water solubility and often aggregate during purification and biophysical investigations. To circumvent this problem many laboratories add oligolysines to the N -and C-termini of peptides that correspond to a TMD. To systematically evaluate the effect of the oligolysines on the biophysical properties of a TMD we synthesized 21 peptides corresponding to either the second (TPIFIINQVSLFLIILHSALYFKY) or sixth (SFHILLIMSSQSLLVPSIIFILAYSLK) TMD of Ste2p, a GPCR from Saccharomyces cerevisiae. Added to the termini of these peptides were either Lys n (n = 1,2,3) or the corresponding native loop residues. The biophysical properties of the peptides were investigated by circular dichroism (CD) spectroscopy in trifluoroethanol-water mixtures, sodium dodecyl sulfate (SDS) micelles and dimyristoylphosphocholine (DMPC)-dimyristoylphosphoglycerol (DMPG) vesicles, and by attenuated total reflection Fourier transform infrared (ATR-FTIR) in DMPC/DMPG multilayers. The results show that the conformation assumed depends on the number of lysine residues and the sequence of the TMD. Identical peptides with native or an equal number of lysine residues exhibited different biophysical properties and structural tendencies.
Structural studies of fragments of G-protein coupled receptors and their ligands by NMR
2009
In the course of my doctoral studies I characterized the structure and dynamics of G-protein coupled receptor (GPCRs) fragments and their ligands by high-resolution NMR. The receptors of the GPCR family are transmembrane proteins of prime biological importance. All members of this family possess similar architecture of seven membrane-spanning-helices and are involved in various signal transduction processes. First part of my work is devoted to the investigation of the structural determinants of the GPCR ligand peptide YY and monitoring the folding process of this peptide in solution. PYY is a 36-residue C-terminally amidated polypeptide that belongs to the neuropeptide Y family of peptide hormones. These molecules are involved in the regulation of a variety of physiological processes, such as for example food uptake. In the second part of my thesis I directed my efforts towards elucidation of the structure and probing the dynamic properties of the transmembrane fragments of the GPCRs in nativelike environments. The subject of my studies was the-factor G-protein coupled Ste2p receptor, which is involved in sensing pheromones in yeast. Two large polypeptide fragments including the first and the second (peptide TM1TM2) and the seventh (peptide TM7) transmembrane domains of the Ste2p receptor were structurally characterized in micellar solution. The obtained results provide important insights into the GPCR architecture in a membrane bilayer. In the first part of my work I focused on the structural determinants and the folding process of the peptide YY (PYY) in solution. Some of the peptides from neuropeptide Y family adopt a well-defined hairpin structure in water that was first shown for avian pancreatic peptide (aPP) using X-ray crystallography. This helical hairpin is commonly referred to as PP-fold and is characterized by a N-terminal polyproline helix, which is back-folded via a-turn onto a C-terminal-helix. The solution structure of the PYY displayed a highly similar helical hairpin, however in the highly homologous neuropeptide Y we were surprised by the absence of the tertiary structure. To investigate the significance of the tertiary contacts, Tyr and Pro residues at the hydrophobic interface of the hairpin-type structure of PYY were replaced by Ala residues, and the conformational and dynamical properties of the resulting peptides were analyzed by high-resolution NMR spectroscopy. Previously we established the 15N1H-NOE as a convenient method to quantify the extent of back-folding. A comparison of the data from different Ala mutant peptides to those of native PYY nicely reflected the differences in backbone rigidity of the N-terminus. Most of the Pro->Ala or the Tyr->Ala mutants possessed increased backbone dynamics, and the differences in N-terminal mobility among them reflected various degrees to which they sample conformations close to the PP-fold. By varying temperature or the methanol content of the aqueous solvent and monitoring chemical shifts we followed the residue-specific formation of tertiary contacts while changing the physical or chemical environment. The PYY peptide in methanol solution was characterized both by determining its solution structure as well as by its internal backbone dynamics as derived from 15N relaxation data. The latter is characterized by a complete loss of tertiary structure. Chemical shifts of C in the heat-denaturation experiments displayed sigmoidal curves with very similar points of inflection indicating that both secondary, as well as tertiary structure in the heat denaturation, was lost synchronously. The results suggest that helical hairpin formation in PYY peptide is both reversible and cooperative and that specific N-and C-terminal tertiary hydrophobic contacts between the polyproline and the-helix promote the folding process. In addition, structural analysis of substitutions in the turn region indicates that the loop does not constrain the hairpin structure. The results may also annimmt, die dem 'PP-fold' ähneln. Durch Variation der Temperatur oder des Methanolgehalts des wässrigen Lösungsmittels und Verfolgung des 'chemical shift' konnten wir die aminosäure-spezifische Bildung der Tertiärkontakte während der Änderung der physikalischen oder chemischen Umgebung verfolgen. Das PYY Peptid in Methanollösung wurde charakterisiert sowohl durch die Bestimmung seiner Lösungsstruktur als auch durch ihre interne 'backbone'-Dynamik mittels 15N-relaxation-Daten. Die 'backbone'-Dynamik zeichnet sich durch einen vollständigen Verlust der tertiären Struktur aus. Die 'Chemical shifts' der C in den Hitze-Denaturierungs-Experimenten zeigten sigmoidale Kurven mit sehr ähnliche Wendepunkten, was darauf hinweist, dass sowohl Sekundär-als auch Tertiärstruktur in der Hitzedenaturierung synchron verloren werden. Die Ergebnisse deuten darauf hin, dass die Bildung des helikalen 'hairpin' im PYY Peptid reversibel und kooperativ ist und dass spezifische N-und C-terminale hydrophobe Tertiärkontakte zwischen der Polyprolinhelix und der-Helix den Faltungsprozess fördern. Darüber hinaus deutet die Strukturanalyse von Substitutionen in der 'turn'-Region darauf hin, dass der 'loop' die 'hairpin'-Struktur nicht hemmt. Die Ergebnisse können auch Auswirkungen für unser Verständnis der Bindung dieser Peptide auf ihren Rezeptoren haben. Im zweiten Teil der Dissertation wurde die Struktur und Dynamik von zwei großen Fragmenten von Ste2p, dem G-Protein-gekoppelten-Faktor-Rezeptor von Hefe untersucht. Beide GPCR-Fragmente wurden exprimiert und aufgereinigt von unseren Kollegen aus der Arbeitsgruppe von Prof. Fred Naider (College of Staten Island, NY). Zuerst untersuchte ich das 73-aminosäure-Peptid TM7 (Ste2p (267-339)) bestehend aus dem dritten extrazellulären 'loop', der siebten Transmembran-Helix und 40 Aminosäuren aus der zytosolische C-terminalen Domäne in Dodecylphosphocholin-Micellen mittels NMR-Spektroskopie. Die Struktur offenbarte die Anwesenheit einer-Helix im Segment von Aminosäurerest 10 bis 30, die um das interne Pro24 gestört wird. 15N-relaxation und RDC-Daten unterstützten einen recht stabilen 'fold' für den Transmembran-Anteil des TM7, hingegen die ausgesetzten Segmente waren flexibler. Die Spin-Label-Daten weisten darauf hin, dass die TM7-Helix in die Dodecylphosphocholin-Micellen integriert wurde, aber zeigten Flexibilität rund um das interne Pro24, da die Aminosäuren 22 bis 26 in die Lösung zeigen, desweiteren zeigten sie einen zweiten Interaktionsort mit der Micelle innerhalb der Region von Aminosäurerest 43 bis 58, die einen Teil einer weniger gut definierten im Entstehen begriffenen Helix bildet. Im weiteren verlängerte ich meine Arbeit an einem einfachen Transmembran-Fragment TM7 zu einem längeren 80-Aminosäure-Doppel-Transmembran-Peptid TM1TM2 (Ste2p (31-110)), bestehend vom 19 Aminosäuren aus der N-terminalen Domäne, die erste Transmembran-Helix, der erste zytoplasmatische 'loop', die zweite Transmembran-Helix und 7 Aminosäuren aus dem ersten extrazellulären 'loop' des Ste2p-Rezeptors. Aufgrund der größeren Komplexität des doppelten Transmembran-Fragments wurden verschiedene Isotopen-Labeling-Muster genutzt: [15N], [15N, 13C], [15N, 13C, 2H]-markiert und selektiv [15N]-markiert an bestimmten Aminosäuren oder protoniert nur an ausgewählten Methyl-Gruppen-Peptiden. Die Struktur des TM 1 TM 2-Peptids in LYSO-palmitoylphosphatidylglycerol-Micellen zeigte das Vorhandensein von drei-Helices, von Aminosäure 39-47, 49-72 und 80-103, mit einer größeren Flexibilität rund um das interne Arg58 der ersten Transmembran-Domäne. Mehrere 'long range-interhelical NOE' Verbindungen unterstützen die Faltung von TM1TM2 in eine Tertiärstruktur, die eine gekreuzte Helix bildet, die sich ausdehnt in Richtung der extrazellulären Regionen und die erhebliche Flexibilität in der G56VRSG60 Region enthält. 15N-relaxation-und Wasserstoff-Deuterium-Austausch-Daten unterstützten einen stabilen 'fold' für die Transmembran-Teile von TM1TM2, während die lösungsmittel-exponierten Segmente flexibler waren. Interessanterweise ist die NMR-Struktur im Einklang mit den Ergebnissen der biochemischen Experimente, die die Ligandenbindungsort in dieser Region des Rezeptors identifizierten. Die erzielten Ergebnisse während meiner Promotionsstudien zeigen wichtige Aspekte der GPCR-Peptid-Liganden PYY-Struktur und seiner Faltung in der Lösung, sowie geben sie Aufschluss über die Struktur der großen Fragmente des Hefe-Pheromon-Rezeptor Ste2p in nativer Micellenumgebung.
Biochemistry, 2000
The Ste2p receptor for R-factor, a tridecapeptide mating pheromone of the yeast Saccharomyces cereVisiae, belongs to the G protein-coupled family of receptors. In this paper we report on the synthesis of peptides corresponding to five of the seven transmembrane domains (M1-M5) and two homologues of the sixth transmembrane domain corresponding to the wild-type sequence and a mutant sequence found in a constitutively active receptor. The secondary structures of all new transmembrane peptides and previously synthesized peptides corresponding to domains 6 and 7 were assessed using a detailed CD analysis in trifluoroethanol, trifluoroethanol-water mixtures, sodium dodecyl sulfate micelles, and dimyristoyl phosphatidyl choline bilayers. Tryptophan fluorescence quenching experiments were used to assess the penetration of the membrane peptides into lipid bilayers. All peptides were predominantly (40-80%) helical in trifluoroethanol and most trifluoroethanol-water mixtures. In contrast, two of the peptides M3-35 (KKKNIIQVLLVASIETSLVFQIKVIFTGDNFKKKG) and M6-31 (KQFDSFHILLIN-leSAQSLLVPSIIFILAYSLK) formed stable-sheet structures in both sodium dodecyl sulfate micelles and DMPC bilayers. Polyacrylamide gel electrophoresis showed that these two peptides formed high molecular aggregates in the presence of SDS whereas all other peptides moved as monomeric species. The peptide (KKKFDSFHILLIMSAQSLLVLSIIFILAYSLKKKS) corresponding to the sequence in the constitutive mutant was predominantly helical under a variety of conditions, whereas the homologous wild-type sequence (KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS) retained a tendency to form-structures. These results demonstrate a connection between a conformational shift in secondary structure, as detected by biophysical techniques, and receptor function. The aggregation of particular transmembrane domains may also reflect a tendency for intermolecular interactions that occur in the membrane environment facilitating formation of receptor dimers or multimers.
Structure of a Double Transmembrane Fragment of a G-Protein-Coupled Receptor in Micelles
Biophysical Journal, 2009
The structure and dynamic properties of an 80-residue fragment of Ste2p, the G-protein-coupled receptor for a-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 [ 15 N], [ 15 N, 13 C], [ 15 N, 13 C, 2 H]-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 a-helices encompassing residues 39-47, 49-72, and 80-103, with higher flexibility around the internal Arg 58 site of TM1. RMSD values of individually superimposed helical segments 39-47, 49-72, and 80-103 were 0.25 5 0.10 Å , 0.40 5 0.13 Å , and 0.57 5 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 G 56 VRSG 60 region. 15 N-relaxation and hydrogendeuterium 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.
Biophysical Journal, 1998
Transmembrane segment (TMS) 7 has been shown to play an important role in the signal transduction function of G-protein-coupled receptors (GPCRs). Although transmembrane segments are most likely to adopt a helical structure, results from a variety of experimental studies involving TMS 7 are inconsistent with it being an ideal ␣-helix. Using results from a search of the structure database and extensive simulated annealing Monte Carlo runs with the new Conformational Memories method, we have identified the conserved (N/D)PxxY region of TMS 7 as the major determinant for deviation of TMS 7 from ideal helicity. The perturbation consists of an Asx turn and a flexible "hinge" region. The Conformational Memories procedure yielded a model structure of TMS 7 which, unlike an ideal ␣-helix, is capable of accommodating all of the experimentally derived geometrical criteria for the interactions of TMS 7 in the transmembrane bundle of GPCRs. In the context of the entire structure of a transmembrane bundle model for the 5HT 2a receptor, the specific perturbation of TMS 7 by the NP sequence suggests a structural hypothesis for the pattern of amino acid conservation observed in TMS 1, 2, and 7 of GPCRs. The structure resulting from the incorporation of the (N/D)P motif satisfies fully the H-bonding capabilities of the 100% conserved polar residues in these TMSs, in agreement with results from mutagenesis experiments. The flexibility introduced by the specific structural perturbation produced by the (NP/DP) motif in TMS 7 is proposed to have a significant role in receptor activation.
Biopolymers, 2012
To conduct biophyiscal analyses on large domains of GPCRs, multi-milligram quantities of highly homogeneous proteins are necessary. This communication discusses the biosynthesis of 4 transmembrane and 5 transmembrane-containing fragments of Ste2p, a GPCR recognizing the Saccharomyces cerevisiae tridecapeptide pheromone α-factor. The target fragments contained the predicted four N-terminal Ste2p[G 31-A 198 ] (4TMN), four C-terminal Ste2p[T 155-L 340 ] (4TMC) or five C-terminal Ste2p[I 120-L 340 ] (5TMC) transmembrane segments of Ste2p. 4TMN was expressed as a fusion protein using a modified pMMHa vector in L-arabinose-induced Escherichia coli BL21-AI, and cleaved with cyanogen bromide. 4TMC and 5TMC were obtained by direct expression using a pET21a vector in IPTG-induced Escherichia coli BL21(DE3) cells. 4TMC and 5TMC were biosynthesized on a preparative scale, isolated in multi-milligram amounts, characterized by MS and investigated by biophysical methods. CD spectroscopy indicated the expected highly α-helical content for 4TMC and 5TMC in membrane mimetic environments. Tryptophan fluorescence showed that 5TMC integrated into the nonpolar region of 1-stearoyl-2hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) micelles. HSQC-TROSY investigations revealed that [ 15 N]-labeled 5TMC in 50% trifluoroethanol-d 2 /H 2 O/0.05% trifluoroacetic acid was stable enough to conduct long multidimensional NMR measurements. The entire Ste2p GPCR was not readily reconstituted from the first two and last five or first three and last four transmembrane domains. Despite their important roles, some aspects of GPCR biology remain under described. Atom-level high resolution information about ligand binding, and ligand-receptor interactions became available only recently 5-20. Nearly all of these studies are on highly
Journal of Biological Chemistry, 2003
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.
Dimers of G-Protein Coupled Receptors as Versatile Storage and Response Units
International Journal of Molecular Sciences, 2014
The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs) are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY) receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY) receptors and could apply to many receptors that use large peptidic agonists.