Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR

Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 04/2004; 101(10):3409-13. DOI: 10.1073/pnas.0308713101
Source: PubMed

ABSTRACT G protein-coupled receptors are cell-surface seven-helical membrane proteins that undergo conformational changes on activation. The mammalian photoreceptor, rhodopsin, is the best-studied member of this superfamily. Here, we provide the first evidence that activation in rhodopsin may involve differential dynamic properties of side-chain versus backbone atoms. High-resolution NMR studies of alpha-(15)N-labeled receptor revealed large backbone motions in the inactive dark state. In contrast, indole side-chain (15)N groups of tryptophans showed well resolved, equally intense NMR signals, suggesting restriction to a single specific conformation.

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Available from: Naveena Yanamala, Mar 12, 2014
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    • "The W6.48 of the CWXP motif in GPCR, which is located at the bottom of the main ligand-binding pocket, is generally expected to function as a key microswitch in GPCR activation16,17. The W6.48 changes position and interaction partners during receptor activation17,18,19. Moreover, the aromatic cluster around W6.48, F[Y]5.47/F[Y]6.52/F[Y]6.51, is proposed to play a role in initiating the receptor activation20. "
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    ABSTRACT: To explore the function of the conserved aromatic cluster F213(5.47), F308(6.51), and F309(6.52) in human β3 adrenergic receptor (hβ3AR). Point mutation technology was used to produce plasmid mutations of hβ3AR. HEK-293 cells were transiently co-transfected with the hβ3AR (wild-type or mutant) plasmids and luciferase reporter vector pCRE-luc. The expression levels of hβ3AR in the cells were determined by Western blot analysis. The constitutive signalling and the signalling induced by the β3AR selective agonist, BRL (BRL37344), were then evaluated. To further explore the interaction mechanism between BRL and β3AR, a three-dimensional complex model of β3AR and BRL was constructed by homology modelling and molecular docking. For F308(6.51), Ala and Leu substitution significantly decreased the constitutive activities of β3AR to approximately 10% of that for the wild-type receptor. However, both the potency and maximal efficacy were unchanged by Ala substitution. In the F308(6.51)L construct, the EC(50) value manifested as a "right shift" of approximately two orders of magnitude with an increased E(max). Impressively, the molecular pharmacological phenotype was similar to the wild-type receptor for the introduction of Tyr at position 308(6.51), though the EC(50) value increased by approximately five-fold for the mutant. For F309(6.52), the constitutive signalling for both F309(6.52)A and F309(6.52)L constructs were strongly impaired. In the F309(6.52)A construct, BRL-stimulated signalling showed a normal E(max) but reduced potency. Leu substitution of F309(6.52) reduced both the E(max) and potency. When F309(6.52) was mutated to Tyr, the constitutive activity was decreased approximately three-fold, and BRL-stimulated signalling was significantly impaired. Furthermore, the double mutant (F308(6.51)A_F309(6.52)A) caused the total loss of β3AR function. The predicted binding mode between β3AR and BRL revealed that both F308(6.51) and F309(6.52) were in the BRL binding pocket of β3AR, while F213(5.47) and W305(6.48) were distant from the binding site. These results revealed that aromatic residues, especially F308(6.51) and F309(6.52), play essential roles in the function of β3AR. Aromatic residues maintained the receptor in a partially activated state and significantly contributed to ligand binding. The results supported the common hypothesis that the aromatic cluster F[Y]5.47/F[Y]6.52/F[Y]6.51 conserved in class A G protein-coupled receptor (GPCR) plays an important role in the structural stability and activation of GPCRs.
    Acta Pharmacologica Sinica 06/2012; 33(8):1062-8. DOI:10.1038/aps.2012.55 · 2.50 Impact Factor
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    • "Unfortunately, a majority of the published methods for obtaining isotopically labeled proteins from eukaryotic expression systems require labor intensive optimization of synthetic media, a problem that is often compounded by low yields. As a result, the use of mammalian expression systems has often been limited to amino-acid type-specific labeling (Arata et al. 1994; Klein-Seetharaman et al. 2002, 2004). A eukaroytic expression system capable of expressing isotopically labeled, well-folded, post-translationally modified proteins at high yield from commercially available media has thus been widely sought. "
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    ABSTRACT: NMR spectroscopic characterization of the structure or the dynamics of proteins generally requires the production of samples isotopically enriched in 15N, 13C, or 2H. The bacterial expression systems currently in use to obtain isotopic enrichment, however, cannot produce a number of eukaryotic proteins, especially those that require post-translational modifications such as N-linked glycosylation for proper folding or activity. Here, we report the use of an adenovirus vector-based mammalian expression system to produce isotopically enriched 15N or 15N/13C samples of an outer domain variant of the HIV-1 gp120 envelope glycoprotein with 15 sites of N-linked glycosylation. Yields for the 15N- and 15N/13C-labeled gp120s after affinity chromatography were 45 and 44 mg/l, respectively, with an average of over 80% isotope incorporation. Recognition of the labeled gp120 by cognate antibodies that recognize complex epitopes showed affinities comparable to the unlabeled protein. NMR spectra, including 1H-15N and 1H-13C HSQCs, 15N-edited NOESY-HSQC, and 3D HNCO, were of high quality, with signal-to-noise consistent with an efficient level of isotope incorporation, and with chemical shift dispersion indicative of a well-folded protein. The exceptional protein yields, good isotope incorporation, and ability to obtain well-folded post-translationally modified proteins make this mammalian system attractive for the production of isotopically enriched eukaryotic proteins for NMR spectroscopy. Electronic supplementary material The online version of this article (doi:10.1007/s10858-011-9506-4) contains supplementary material, which is available to authorized users.
    Journal of Biomolecular NMR 06/2011; 50(3):197-207. DOI:10.1007/s10858-011-9506-4 · 3.31 Impact Factor
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    • "Ligand binding motifs and pharmacological responses for the CB1 receptor and other GPCRs have been attributed to specific GPCR TM helix conformations and orientations within the cell membrane bilayer [19] [20]. Conformational exchange in the micro-millisecond regime is important to the binding and pharmacodynamics of GPCR ligands, and helices 7 and 8 are considered critical to the stability of the GPCR-G-protein interaction at the cytoplasmic C-terminus [21]. Thus, information on GPCR TM helix structure and orientation appears essential to understanding the (patho)physiological roles of endogenous cannabinoids and elucidating the pharmacophore requirements for synthetic CB1-receptor ligands as potential drugs. "
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    ABSTRACT: Little direct information is available regarding the influence of membrane environment on transmembrane (TM) G-protein-coupled receptor (GPCR) conformation and dynamics. The human CB1 cannabinoid receptor (hCB1) is a prominent GPCR pharmacotherapeutic target in which helix 7 appears critical to ligand recognition. We have chemically synthesized a hCB1 peptide corresponding to a segment of TM helix 7 and the entire contiguous helix 8 domain (fourth cytoplasmic loop) and reconstituted it in defined phospholipid-bilayer model membranes. Using an NMR-based strategy combined with molecular dynamics simulations, we provide the first direct experimental description of the orientation of hCB1 helix 7 in phospholipid membranes of varying thickness and the mechanism by which helix-7 conformation adjusts to avoid hydrophobic mismatch. Solid-state 15N NMR data show that hCB1 helices 7 and 8 reconstituted into phospholipid bilayers are oriented in a TM and in-plane (i.e., parallel to the phospholipid membrane surface) fashion, respectively. TM helix orientation is influenced by the thickness of the hydrophobic membrane bilayer as well as the interaction of helix 8 with phospholipid polar headgroups. Molecular dynamics simulations show that a decrease in phospholipid chain-length induces a kink at P394 in TM helix 7 to avoid hydrophobic mismatch. Thus, the NP(X)nY motif found in hCB1 and highly conserved throughout the GPCR superfamily is important for flexing helix 7 to accommodate bilayer thickness. Dynamic modulation of hCB1-receptor TM helix conformation by its membrane environment may have general relevance to GPCR structure and function.
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