Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

Wiktor, Maciej; Morin, Sébastien; Kebbel, Fabian; Grzesiek, Stephan

doi:10.1007/s10858-012-9688-4

Cited by 15 publications

(16 citation statements)

References 69 publications

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“…Consequently, 1 H, 15 N HSQC spectra of GPCRs in solution show relatively large linewidths. [43,44] In contrast, 1 H, 13 C correlation experiments show a somewhat better resolution and display quite a number of well-resolved cross-peaks, which most likely come from the highly mobile termini of the receptor. [4] Apparently, the axially symmetric rotations along with the segmental mobility of the receptor provide a significant reduction in the dipolar couplings along with a sufficient increase in relaxation times.…”

Section: Discussionmentioning

confidence: 96%

The G‐Protein‐Coupled Neuropeptide Y Receptor Type 2 is Highly Dynamic in Lipid Membranes as Revealed by Solid‐State NMR Spectroscopy

Schmidt

Thomas

Müller

et al. 2014

Chemistry A European J

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In spite of the recent success in crystallizing several G-protein-coupled receptors (GPCRs), a comprehensive biophysical characterization of these molecules under physiological conditions also requires the study of the molecular dynamics of these proteins. The molecular mobility of the human neuropeptide Y receptor type 2 reconstituted into dimyristoylphosphatidylcholine (DMPC) membranes was investigated by means of solid-state NMR spectroscopy. Static (15) N NMR spectra show that the receptor performs axially symmetric motions in the membrane, and several residues undergo large amplitude fluctuations. This was confirmed by quantitative measurements of the motional (1) H,(13) C order parameter of the CH, CH2 , and CH3 groups. In directly polarized (13) C NMR experiments, these order parameters showed astonishingly low values of SCH =0.55, S CH 2=0.33, and S CH 3=0.17, which corresponds to segmental amplitudes of approximately 50° in the backbone and approximately 50-60° in the side chain. At physiological temperature, (2) H NMR spectra of the deuterated receptor showed a narrow component that is indicative of molecular order parameters of S≤0.3 superimposed with a very broad spectrum that could stem from the transmembrane α-helices. These results suggest that the crystal structures of GPCRs only represent a static snapshot of these highly mobile molecules, which undergo significant structural fluctuations with relatively large amplitudes in a liquid-crystalline membrane at physiological temperature.

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Section: Discussionmentioning

confidence: 96%

The G‐Protein‐Coupled Neuropeptide Y Receptor Type 2 is Highly Dynamic in Lipid Membranes as Revealed by Solid‐State NMR Spectroscopy

Schmidt

Thomas

Müller

et al. 2014

Chemistry A European J

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“…Some cysteines in the helical domains and in the C-terminus of the GPCRs are not well-protected in the micelle and may induce formation of non-native oligomers. It has been shown for several GPCRs that reducing the number of cysteine residues in the receptor sequence by mutations improves sample quality (Li et al , 2008 ;Wiktor et al , 2013 ).…”

Section: Discussionmentioning

confidence: 99%

“…Another important issue for the stability of in vitro preparations of GPCRs is the cysteine bond pattern, as it has been shown for the chemokine receptor CCR5 (Wiktor et al , 2013 ). Most GPCRs contain a highly conserved disulfide bridge between the third helix and the second extracellular loop (Kolakowski , Jr., 1994 ).…”

Section: Introductionmentioning

confidence: 99%

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Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration

Witte

Kaiser

Schmidt

et al. 2013

Biological Chemistry

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In vitro folding of G protein-coupled receptors into a detergent environment represents a promising strategy for obtaining sufficient amounts of functional receptor molecules for structural studies. Typically, these preparations exhibit a poor long-term stability especially at the required high protein concentration. Here, we report a protocol for the stabilization of the Escherichia coli-expressed and subsequently folded neuropeptide Y receptor type 2. We identified the free cysteines in the receptor as one major reason for intermolecular protein aggregation. Therefore, six out of the eight cysteine residues were mutated to alanine or serine without any significant loss of functionality of the receptor as demonstrated in cell culture models. Furthermore, the disulfide bond between the remaining two cysteines was irreversibly formed by applying oxidative in vitro folding. Applying this strategy, the stability of the functionally folded Y2 receptor could be increased to 20 days at a concentration of 15 μm in a micelle environment consisting of 1,2-diheptanoyl-sn-glycero-3-phosphocholine and n-dodecyl-ß-D-maltoside.

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“…However, large membrane protein complexes rotate slowly in the solvent, which leads to anisotropic samples. Signals obtained from these samples show broad line widths, which limits the application of solution NMR to GPCR complexes [ 142 , 143 , 144 , 145 ]. Moreover, the signals of the bound ligand appear broadened and eventually become undetectable when the ligand exchanges between bound and unbound states slowly, with rates of ~1 ms −1 (exchange broadening, reviewed in [ 146 ]), which is typical for high-affinity peptide ligands.…”

Section: A Ligand’s Perspectivementioning

confidence: 99%

Capturing Peptide–GPCR Interactions and Their Dynamics

Kaiser

Coin

2020

Molecules

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Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes.

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Biophysical and structural investigation of bacterially expressed and engineered CCR5, a G protein-coupled receptor

Cited by 15 publications

References 69 publications

The G‐Protein‐Coupled Neuropeptide Y Receptor Type 2 is Highly Dynamic in Lipid Membranes as Revealed by Solid‐State NMR Spectroscopy

The G‐Protein‐Coupled Neuropeptide Y Receptor Type 2 is Highly Dynamic in Lipid Membranes as Revealed by Solid‐State NMR Spectroscopy

Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration

Capturing Peptide–GPCR Interactions and Their Dynamics

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