Amino acid conservation and interactions in rhodopsin: Probing receptor activation by NMR spectroscopy

Pope, Andreyah; Eilers, Markus; Reeves, Philip J.; Smith, Steven O.

doi:10.1016/j.bbabio.2013.10.007

Cited by 17 publications

(23 citation statements)

References 91 publications

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“…Spectroscopic evidence suggested changes in the environment of W 6.48 upon rhodopsin activation3132. However, a rotamer change of W 6.48 was not observed in any crystal structure of active GPCRs578933, but only found in the structures of active-intermediate NTSR1-ELF and NTSR1-LF18.…”

Section: Resultsmentioning

confidence: 97%

Structure and dynamics of a constitutively active neurotensin receptor

Krumm

Lee

Bhattacharya

et al. 2016

Sci Rep

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Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.

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Section: Resultsmentioning

confidence: 97%

Structure and dynamics of a constitutively active neurotensin receptor

Krumm

Lee

Bhattacharya

et al. 2016

Sci Rep

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“…Following significant expansions in genome resources over recent years, the N83 substitution appears to have convergently evolved in an ever more scattered collection of vertebrate rhodopsins, with no obvious correlations to life history traits . Furthermore, studies that emphasize the multi‐faceted structural roles of this site make it clear that resolving the functional mechanisms of the N83 substitution, and its relationship to organism lifestyle, will depend on better understanding its interactions with other sites.…”

Section: Discussionmentioning

confidence: 99%

A comparative study of rhodopsin function in the great bowerbird (Ptilonorhynchus nuchalis): Spectral tuning and light‐activated kinetics

et al. 2016

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Rhodopsin is the visual pigment responsible for initiating the phototransduction cascade in vertebrate rod photoreceptors. Although well-characterized in a few model systems, comparative studies of rhodopsin function, particularly for nonmammalian vertebrates are comparatively lacking. Bowerbirds are rare among passerines in possessing a key substitution, D83N, at a site that is otherwise highly conserved among G protein-coupled receptors. While this substitution is present in some dim-light adapted vertebrates, often accompanying another unusual substitution, A292S, its functional relevance in birds is uncertain. To investigate functional effects associated with these two substitutions, we use the rhodopsin gene from the great bowerbird (Ptilonorhynchus nuchalis) as a background for site-directed mutagenesis, in vitro expression and functional characterization. We also mutated these sites in two additional rhodopsins that do not naturally possess N83, chicken and bovine, for comparison. Both sites were found to contribute to spectral blue-shifts, but had opposing effects on kinetic rates. Substitutions at site 83 were found to primarily affect the kinetics of light-activated rhodopsin, while substitutions at site 292 had a larger impact on spectral tuning. The contribution of substitutions at site 83 to spectral tuning in particular depended on genetic background, but overall, the effects of substitutions were otherwise surprisingly additive, and the magnitudes of functional shifts were roughly similar across all three genetic backgrounds. By employing a comparative approach with multiple species, our study provides new insight into the joint impact of sites 83 and 292 on rhodopsin structure-function as well as their evolutionary significance for dim-light vision across vertebrates.

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“…The largest body of data is available for rhodopsin, for which the ground and activated states have been characterized. [22][23][24] Also, the full structure of the chemokine receptor CXCR1 has recently been solved by solid-state NMR spectroscopy. [4] Furthermore, the conformations of the peptides neurotensin [25] and bradykinin [26] bound to their GPCRs have been determined by this method.…”

Section: Introductionmentioning

confidence: 99%

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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Amino acid conservation and interactions in rhodopsin: Probing receptor activation by NMR spectroscopy

Cited by 17 publications

References 91 publications

Structure and dynamics of a constitutively active neurotensin receptor

Structure and dynamics of a constitutively active neurotensin receptor

A comparative study of rhodopsin function in the great bowerbird (Ptilonorhynchus nuchalis): Spectral tuning and light‐activated kinetics

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

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