Studies of the Structure of the N‐Terminal Domain from the Y4 Receptor—a G Protein‐Coupled Receptor—and its Interaction with Hormones from the NPY Family

Zou, Chao; Kumaran, Sowmini; Marković, Stefan; Walser, Reto; Zerbe, Oliver

doi:10.1002/cbic.200800221

Cited by 20 publications

(27 citation statements)

References 53 publications

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“…A short helix in the extracellular segment from Phe38 to Val45 of the N-terminus is also visible in all fragments. This helix is likely surface-associated and was also observed in an N-terminal fragment of the NPY4 GPCR (39). A region in the loop between TM2 and the third helix in both TM123 and TM127 displays some disposition to form a helical secondary structure.…”

Section: Conformational Preferencesmentioning

confidence: 78%

NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates

Poms

Ansorge

Martínez-Gil

et al. 2016

Journal of Biological Chemistry

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Folding of G-protein coupled receptors (GPCRs) according to the two-stage model (Popot et al., Biochemistry 29(1990), 4031) is postulated to proceed in 2 steps: Partitioning of the polypeptide into the membrane followed by diffusion until native contacts are formed. Herein we investigate conformational preferences of fragments of the yeast Ste2p receptor using NMR. Constructs comprising the first, the first two and the first three transmembrane (TM) segments, as well as a construct comprising TM1-TM2 covalently linked to TM7 were examined. We observed that the isolated TM1 does not form a stable helix nor does it integrate well into the micelle. TM1 is significantly stabilized upon interaction with TM2, forming a helical hairpin reported previously (Neumoin et al., Biophys. J. 96(2009), 3187), and in this case the protein integrates into the hydrophobic interior of the micelle. TM123 displays a strong tendency to oligomerize, but hydrogen exchange data reveal that the center of TM3 is solvent exposed. In all GPCRs so-far structurally characterized TM7 forms many contacts with TM1 and TM2. In our study TM127 integrates well into the hydrophobic environment, but TM7 does not stably pack against the remaining helices. Topology mapping in microsomal membranes also indicates that TM1 does not integrate in a membrane-spanning fashion, but that TM12, TM123 and TM127 adopt predominantly nativelike topologies. The data from our study would be consistent with the retention of individual helices of incompletely synthesized GPCRs in the vicinity of the translocon until the complete receptor is released into the membrane interior. (7), and the structure of a GPCRarrestin complex was solved (8).While our knowledge of the structure of GPCRs in various states and their mode of activation and desensitization is rapidly increasing, detailed information on their folding pathways is still lacking. The popular refined two-stage model from Popot and Engelman postulates that secondary structure forms when the peptide chain partitions into the membranewater interface (9-11). However, proteins destined for membrane insertion are generally subjected to the concerted action of translating ribosomes in the cytoplasm and translocon complexes located in the endoplasmic reticulum (ER) of eukaryotes or in the plasma membrane of bacteria (12). In order to traffic proteins to membranes in most cells the signal-recognition particle targets the nascent chains emerging from the ribosome tunnel to the translocon complex (13 (12,(18)(19)(20). Folding of polytopic membrane proteins is the result of a series of events that include helixinsertion into the hydrophobic core and sequestering of loop-sequences into cytosolic or extracellular space (11,21). The timing of the chain insertion and the localization of TM helices would be expected to be a consequence of the amino acid sequence and the interaction of the growing polypeptide chain with the membrane. Recently, however, the first evidence was obtained that helices might change their locat...

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Section: Conformational Preferencesmentioning

confidence: 78%

NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates

Poms

Ansorge

Martínez-Gil

et al. 2016

Journal of Biological Chemistry

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“…[13] Numerous studies by us during the last years have indicated that fragments corresponding to one or more TM helices do form stable secondary structure. [14][15][16][17][18] We have learned during those studies that the stability very much depends on the presence of polar residues in central regions of the TM helices. The exact structure of the TM helices in addition depends on tertiary contacts, and those may be missing in the segment.…”

Section: Studies Of 2-3 Tm Helix Fragments From the Y4 Receptormentioning

confidence: 99%

“…[22] To probe for their conformational preferences and their possible interactions with the ligands, which are peptide hormones of the NPY family, we have produced them in 15 N-labelled form. [16,17] H]-HSQC spectra revealed them all to be largely unfolded with the exception of the N-terminal domain from the Y4 receptor. [17] This peptide contains two α-helical stretches at the termini, separated by a long and flexible loop.…”

Section: Investigations Of the Free N-terminal Domains Of The Y4 Recementioning

confidence: 99%

“…[17] This peptide contains two α-helical stretches at the termini, separated by a long and flexible loop. [16] Interestingly, using surface-plasmon resonance (SPR), we could detect a weak (Kd of approx. 50 µM) binding to the pancreatic polypeptide (PP), which is the peptide that associates with the Y4 receptor with the highest affinity.…”

Section: Investigations Of the Free N-terminal Domains Of The Y4 Recementioning

confidence: 99%

“…To be honest: The study of fragments is heavily under debate since we are investi-N-terminal domain and anionic from the ligand PP. [16] Considering that peptides from the NPY family associate with the membrane we speculate that a transient interaction of the peptides with the N-terminal domain might help to transfer them into the receptor binding-pocket (Fig. 2).…”

Section: Introductionmentioning

confidence: 99%

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Approaches towards Structures of Y Receptors, Examples of Human G-Protein Coupled Receptors, by Solution NMR

Walser

Zerbe²

2012

Chimia

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Despite recent advances no solution structure for a true G-protein coupled receptor (GPCR) is available today due to biochemical and spectroscopic problems. Herein we review our attempts to obtain assignments of GPCRs based on fragments comprising 2-3 transmembrane helices. The fragments are expressed in a heterologous system, and studied in detergent micelles using solution NMR spectroscopy. We report on the status of assignments of fragments from the Y4 receptor, a human GPCR. Assignments for the majority of the backbone resonances are available as well as sidechain assignments for the first two TM helices. Residues of TM4 are largely invisible. We review technical issues in preparing these samples and in the data analysis. In addition we developed an approach in which we have grafted the extracellular loops of the Y1 receptor onto a beta-barrel scaffold derived from the E. coli outer membrane protein OmpA. We could demonstrate that all loops can be successfully transferred, and that the resulting protein can be successfully refolded. The system is capable of recognizing the ligands from the neuropeptide Y family. Abstract: Despite recent advances no solution structure for a true G-protein coupled receptor (GPCR) is available today due to biochemical and spectroscopic problems. Herein we review our attempts to obtain assignments of GPCRs based on fragments comprising 2-3 transmembrane helices. The fragments are expressed in a heterologous system, and studied in detergent micelles using solution NMR spectroscopy. We report on the status of assignments of fragments from the Y4 receptor, a human GPCR. Assignments for the majority of the backbone resonances are available as well as sidechain assignments for the first two TM helices. Residues of TM4 are largely invisible. We review technical issues in preparing these samples and in the data analysis. In addition we developed an approach in which we have grafted the extracellular loops of the Y1 receptor onto a beta-barrel scaffold derived from the E. coli outer membrane protein OmpA. We could demonstrate that all loops can be successfully transferred, and that the resulting protein can be successfully refolded. The system is capable of recognizing the ligands from the neuropeptide Y family.

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Invited review GPCR structural characterization: Using fragments as building blocks to determine a complete structure

et al. 2014

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The structural characterization of G protein-coupled receptors has surged since the development of methodologies to facilitate the crystallization of these highly helical, seven transmembrane, integral membrane receptors. In the past seven years, eighteen GPCR structures were determined by X-ray crystallography. The crystal structures represent a static picture of these conformationally flexible signal transducers. Analyses that probe their dynamics and conformational changes require other techniques, in particular solution state nuclear magnetic resonance studies. Such investigations are challenged by the size of GPCRs, their α-helical structure, which limits resonance dispersion, their tendencies to aggregate in micellar preparations and their conformational heterogeneity. For many years, groups have been studying GPCR fragments as a means to overcome some of these difficulties. The results of these fragment analyses are presented here. Review of the literature reveals that much of the original work depended on circular dichroism, infra-red spectroscopy and fluorescence approaches. High resolution structures obtained by NMR are compared, where applicable, to the available crystal structures. In most cases, the work done on fragments by biophysical analysis is validated by these comparisons. Our perspective on the field of GPCR fragment analysis is presented together with the future goals that must be considered if work with fragments is continued.

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Studies of the Structure of the N‐Terminal Domain from the Y4 Receptor—a G Protein‐Coupled Receptor—and its Interaction with Hormones from the NPY Family

Cited by 20 publications

References 53 publications

NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates

NMR Investigation of Structures of G-protein Coupled Receptor Folding Intermediates

Approaches towards Structures of Y Receptors, Examples of Human G-Protein Coupled Receptors, by Solution NMR

Invited review GPCR structural characterization: Using fragments as building blocks to determine a complete structure

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