NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates (original) (raw)
Related papers
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.
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 Peptide Science, 2015
This report summarizes recent biophysical and protein expression experiments on polypeptides containing the N-terminus, the first, second and third transmembrane domains and the contiguous loops of the-factor receptor Ste2p, a G protein-coupled receptor. The 131-residue polypeptide Ste2p(G31-R161), TM1-TM3 was investigated by solution NMR in trifluorethanol/water: TM1-TM3 contains helical transmembrane domains at the predicted locations, supported by continuous sets of medium-range NOEs. In addition, a short helix N-terminal to TM1 was detected, as well as a short helical stretch in the first extracellular loop. Two 161-residue polypeptides, [Ste2p(M1-R161), NT-TM1-TM3], that contain the entire N-terminal sequence, one with a single mutation, were directly expressed and isolated from E. coli in yields as high as 30 mg/L. Based on its increased stability, the L11P mutant will be used in future experiments to determine long-range interactions. The study demonstrated that 3-TM domains of a yeast GPCR can be produced in isotopically labeled form suitable for solution NMR studies. The quality of spectra is superior to data recorded in micelles and allows more rapid data analysis. No tertiary contacts have been determined, and if present, they are likely transient. This observation supports earlier studies by us that secondary structure was retained in smaller fragments, both in organic solvents and in detergent micelles, but that stable tertiary contacts may only be present when the protein is imbedded in lipids.
Advances in experimental medicine and biology, 2014
Experimental observations of the dependence of function and organization of G protein-coupled receptors (GPCRs) on their lipid environment have stimulated new quantitative studies of the coupling between the proteins and the membrane. It is important to develop such a quantitative understanding at the molecular level because the effects of the coupling are seen to be physiologically and clinically significant. Here we review findings that offer insight into how membrane-GPCR coupling is connected to the structural characteristics of the GPCR, from sequence to 3D structural detail, and how this coupling is involved in the actions of ligands on the receptor. The application of a recently developed computational approach designed for quantitative evaluation of membrane remodeling and the energetics of membrane-protein interactions brings to light the importance of the radial asymmetry of the membrane-facing surface of GPCRs in their interaction with the surrounding membrane. As the rad...
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, 2002
A major, unresolved question in signal transduction by G protein coupled receptors (GPCRs) is to understand how, at atomic resolution, a GPCR activates a G protein. A step toward answering this question was made with the determination of the high-resolution structure of rhodopsin; we now know the intramolecular interactions that characterize the resting conformation of a GPCR. To what degree does this structure represent a structural paradigm for other GPCRs, especially at the cytoplasmic surface where GPCR-G protein interaction occurs and where the sequence homology is low among GPCRs? To address this question, we performed NMR studies on ∼35-residue-long peptides including the critical second intracellular loop (i2) of the R2A adrenergic receptor (AR) and of rhodopsin. To stabilize the secondary structure of the peptide termini, 4-12 residues from the adjacent transmembrane helices were included and structures determined in dodecylphosphocholine micelles. We also characterized the effects on an R2A AR peptide of a D130I mutation in the conserved DRY motif. Our results show that in contrast to the L-shaped loop in the i2 of rhodopsin, the i2 of the R2A AR is predominantly helical, supporting the hypothesis that there is structural diversity within GPCR intracellular loops. The D130I mutation subtly modulates the helical structure. The spacing of nonpolar residues in i2 with helical periodicity is a predictor of helical versus loop structure. These data should lead to more accurate models of the intracellular surface of GPCRs and of receptor-mediated G protein activation. G protein coupled receptors (GPCRs) 1 comprise a large and diverse family of receptors, each of which contains seven transmembrane (TM) spanning helices and activates an associated heterotrimeric G protein. Recently, Palczewski et al. solved the first high-resolution structure of a prototypical GPCR, rhodopsin (1), which activates the G protein transducin. This structure provided an important piece to the puzzle of GPCR-mediated signal transduction. While this accomplishment represents a large step forward on the path toward a complete understanding of GPCR mechanisms, this lone structure of an inactive-state GPCR leaves unanswered the question of how rhodopsin and other GPCRs actually activate their G proteins. The laboratory of Khorana (2, 3) and others (4) have recently provided information on the conformational changes that occur during activation and the molecular interactions that form between rhodopsin and transducin. Rhodopsin has been the focus of GPCR biophysical studies because of the ability to obtain working quantities of the purified, stable, nonaggregated receptor. However, the applicability of the rhodopsin paradigm to other GPCRs is a matter of speculation (5). The seven TM helices contain several residues that are highly conserved among rhodopsinlike GPCRs (6), and, thus, the seven-TM bundle of rhodopsin likely provides a good three-dimensional template for other GPCRs (7). However, in the intracellular loops, there are very few conserved residues (6). Together with the considerable variability in length of the third intracellular loop (8), this suggests that the intracellular surface of GPCRs, which forms the GPCR-G protein interface, may be structurally dissimilar. Despite this possibility, investigators often utilize rhodopsin structural data to model the intracellular surface
G-protein Coupled Receptor Dimerization
Iranian Journal of Pharmacology Therapeutics, 2003
A growing body of evidence suggests that GPCRs exist and function as dimers or higher oligomers. The evidence for GPCR dimerization comes from biochemical, biophysical and functional studies. In addition, researchers have shown the occurrence of heterodimerization between different members of the GPCR family. Two receptors can interact with each other to make a dimer through their extracellular loops, transmembrane helices and intracellular loops. The nature of bonds between two receptors can vary from covalent (e.g. disulphide bonds) to non-covalent (for instance hydrophobic interactions between transmembrane helices or coiled coil structures) or a combination of both. Dimerization can occur in and affect different stages of a receptor's life, namely trafficking, signaling and internalization, and can be seen as the natural way to regulate receptor activity or increase the functional repertoire of proteins. Different structures for GPCR dimers have been proposed, for example a simple contact dimer or an interlocking domain-swapped structure. Here we introduce some of the information available on GPCR dimerization, which includes early studies that had been dismissed until the relatively recent past and some of the more recent data which has vindicated these early studies
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.