Structures of the Rhodopsin-Transducin Complex: Insights into G-Protein Activation.
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ABSTRACT:
SUBMITTER: Gao Y
PROVIDER: S-EPMC6707884 | BioStudies | 2019-01-01
REPOSITORIES: biostudies
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Project description:Upon illumination the visual receptor rhodopsin (Rho) transitions to the activated form Rho(?), which binds the heterotrimeric G protein, transducin (Gt) causing GDP to GTP exchange and Gt dissociation. Using succinylated concanavalin A (sConA) as a probe, we visualized native Rho dimers solubilized in 1mM n-dodecyl-?-d-maltoside (DDM) and Rho monomers in 5mM DDM. By nucleotide depletion and affinity chromatography together with crosslinking and size exclusion chromatography, we trapped and purified nucleotide-free Rho(?)·Gt and sConA-Rho(?)·Gt complexes kept in solution by either DDM or lauryl-maltose-neopentyl-glycol (LMNG). The 3 D envelope calculated from projections of negatively stained Rho(?)·Gt-LMNG complexes accommodated two Rho molecules, one Gt heterotrimer and a detergent belt. Visualization of triple sConA-Rho(?)·Gt complexes unequivocally demonstrated a pentameric assembly of the Rho(?)·Gt complex in which the photoactivated Rho(?) dimer serves as a platform for binding the Gt heterotrimer. Importantly, individual monomers of the Rho(?) dimer in the heteropentameric complex exhibited different capabilities for regeneration with either 11-cis or 9-cis-retinal.
2013-01-01 | S-EPMC3633645 | BioStudies
Project description:Heterotrimeric G proteins become activated after they form a catalytically active complex with activated G protein-coupled receptors (GPCRs) and GTP replaces GDP on the G protein alpha-subunit. This transient coupling can be stabilized by nucleotide depletion, resulting in an empty-nucleotide G protein-GPCR complex. Efficient and reproducible formation of conformationally homogeneous GPCR-Gt complexes is a prerequisite for structural studies. Herein, we report isolation conditions that enhance the stability and preserve the activity and proper stoichiometry of productive complexes between the purified prototypical GPCR, rhodopsin (Rho), and the rod cell-specific G protein, transducin (Gt). Binding of purified Gt to photoactivated Rho (Rho*) in n-dodecyl beta-D-maltoside (DDM) examined by gel filtration chromatography was generally modest, and purified complexes provided heterogeneous ratios of protein components, most likely because of excess detergent. Rho*-Gt complex stability and activity were greatly increased by addition of phospholipids such as DOPC, DOPE, and DOPS and asolectin to detergent-containing solutions of these proteins. In contrast, native Rho*-Gt complexes purified directly from light-exposed bovine ROS membranes by sucrose gradient centrifugation exhibited improved stability and the expected 2:1 stoichiometry between Rho* and Gt. These results strongly indicate a lipid requirement for stable complex formation in which the likely oligomeric structure of Rho provides a superior platform for coupling to Gt, and phospholipids likely form a matrix to which Gt can anchor through its myristoyl and farnesyl groups. Our findings also demonstrate that the choice of detergent and purification method is critical for obtaining highly purified, stable, and active complexes with appropriate stoichiometry between GPCRs and G proteins needed for structural studies.
2009-01-01 | S-EPMC2753473 | BioStudies
Project description:G protein-coupled receptor (GPCR) signaling is crucial for many physiological processes. A signature of such pathways is high amplification, a concept originating from retinal rod phototransduction, whereby one photoactivated rhodopsin molecule (Rho*) was long reported to activate several hundred transducins (GT*s), each then activating a cGMP-phosphodiesterase catalytic subunit (GT*·PDE*). This high gain at the Rho*-to-GT* step has been challenged more recently, but estimates remain dispersed and rely on some nonintact rod measurements. With two independent approaches, one with an extremely inefficient mutant rhodopsin and the other with WT bleached rhodopsin, which has exceedingly weak constitutive activity in darkness, we obtained an estimate for the electrical effect from a single GT*·PDE* molecular complex in intact mouse rods. Comparing the single-GT*·PDE* effect to the WT single-photon response, both in Gcaps -/- background, gives an effective gain of only ∼12-14 GT*·PDE*s produced per Rho*. Our findings have finally dispelled the entrenched concept of very high gain at the receptor-to-G protein/effector step in GPCR systems.
2019-01-01 | S-EPMC6421417 | BioStudies
Project description:Visual signal transduction serves as one of the best understood G protein-coupled receptor signaling systems. Signaling is initiated when a photon strikes rhodopsin (Rho) causing a conformational change leading to productive interaction of this G protein-coupled receptor with the heterotrimeric G protein, transducin (Gt). Here we describe a new method for Gt purification from native bovine rod photoreceptor membranes without subunit dissociation caused by exposure to photoactivated rhodopsin (Rho*). Native electrophoresis followed by immunoblotting revealed that Gt purified by this method formed more stable heterotrimers and interacted more efficiently with membranes containing Rho* or its target, phosphodiesterase 6, than did Gt purified by a traditional method involving subunit dissociation and reconstitution in solution without membranes. Because these differences could result from selective extraction, we characterized the type and amount of posttranslational modifications on both purified native and reconstituted Gt preparations. Similar N-terminal acylation of the Gtalpha subunit was observed for both proteins as was farnesylation and methylation of the terminal Gtgamma subunit Cys residue. However, hydrogen/deuterium exchange experiments revealed less incorporation of deuterium into the Gtalpha and Gtbeta subunits of native Gt as compared to reconstituted Gt. These findings may indicate differences in conformation and heterotrimer complex formation between the two preparations or altered stability of the reconstituted Gt that assembles differently than the native protein. Therefore, Gt extracted and purified without subunit dissociation appears to be more appropriate for future studies.
2008-01-01 | S-EPMC2645919 | BioStudies
Project description:The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (GT). This results in the dissociation of GT into its component ?T-GTP and ?1?1 subunit complex. Structural information for the Rho*-GT complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (GT*) comprising a G?T/G?i1 chimera (?T*) and ?1?1 The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to G?T* is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one GT*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the ?2-adrenergic receptor-GS complex, including a flexible ?T* helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex.
2017-01-01 | S-EPMC5572916 | BioStudies
Project description:The process of vision is initiated when the G protein-coupled receptor, rhodopsin (Rho), absorbs a photon and transitions to its activated Rho(?) form. Rho(?) binds the heterotrimeric G protein, transducin (G(t)) inducing GDP to GTP exchange and G(t) dissociation. Using nucleotide depletion and affinity chromatography, we trapped and purified the resulting nucleotide-free Rho(?)·G(t) complex. Quantitative SDS-PAGE suggested a 2:1 molar ratio of Rho(?) to G(t) in the complex and its mass determined by scanning transmission electron microscopy was 221±12kDa. A 21.6Å structure was calculated from projections of negatively stained Rho(?)·G(t) complexes. The molecular envelope thus determined accommodated two Rho molecules together with one G(t) heterotrimer, corroborating the heteropentameric structure of the Rho(?)·G(t) complex.
2011-01-01 | S-EPMC3210324 | BioStudies
Project description:A novel combination of experimental data and extensive computational modeling was used to explore probable protein-protein interactions between photoactivated rhodopsin (R*) and experimentally determined R*-bound structures of the C-terminal fragment of alpha-transducin (Gt(alpha)(340-350)) and its analogs. Rather than using one set of loop structures derived from the dark-adapted rhodopsin state, R* was modeled in this study using various energetically feasible sets of intracellular loop (IC loop) conformations proposed previously in another study. The R*-bound conformation of Gt(alpha)(340-350) and several analogs were modeled using experimental transferred nuclear Overhauser effect data derived upon binding R*. Gt(alpha)(340-350) and its analogs were docked to various conformations of the intracellular loops, followed by optimization of side-chain spatial positions in both R* and Gt(alpha)(340-350) to obtain low-energy complexes. Finally, the structures of each complex were subjected to energy minimization using the OPLS/GBSA force field. The resulting residue-residue contacts at the interface between R* and Gt(alpha)(340-350) were validated by comparison with available experimental data, primarily from mutational studies. Computational modeling performed for Gt(alpha)(340-350) and its analogs when bound to R* revealed a consensus of general residue-residue interactions, necessary for efficient complex formation between R* and its Gt(alpha) recognition motif.
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Project description:Signaling of the prototypical G protein-coupled receptor (GPCR) rhodopsin through its cognate G protein transducin (Gt) is quenched when arrestin binds to the activated receptor. Although the overall architecture of the rhodopsin/arrestin complex is known, many questions regarding its specificity remain unresolved. Here, using FTIR difference spectroscopy and a dual pH/peptide titration assay, we show that rhodopsin maintains certain flexibility upon binding the "finger loop" of visual arrestin (prepared as synthetic peptide ArrFL-1). We found that two distinct complexes can be stabilized depending on the protonation state of E3.49 in the conserved (D)ERY motif. Both complexes exhibit different interaction modes and affinities of ArrFL-1 binding. The plasticity of the receptor within the rhodopsin/ArrFL-1 complex stands in contrast to the complex with the C terminus of the Gt ?-subunit (G?CT), which stabilizes only one specific substate out of the conformational ensemble. However, Gt ?-subunit binding and both ArrFL-1-binding modes involve a direct interaction to conserved R3.50, as determined by site-directed mutagenesis. Our findings highlight the importance of receptor conformational flexibility and cytoplasmic proton uptake for modulation of rhodopsin signaling and thereby extend the picture provided by crystal structures of the rhodopsin/arrestin and rhodopsin/ArrFL-1 complexes. Furthermore, the two binding modes of ArrFL-1 identified here involve motifs of conserved amino acids, which indicates that our results may have elucidated a common modulation mechanism of class A GPCR-G protein/-arrestin signaling.
2018-01-01 | S-EPMC5868258 | BioStudies
Project description:Transducin mediates signal transduction in a classical G protein-coupled receptor (GPCR) phototransduction cascade. Interactions of transducin with the receptor and the effector molecules had been extensively investigated and are currently defined at the atomic level. However, partners and functions of rod transducin ? (G?t 1) and ?? (G?1?1) outside the visual pathway are not well-understood. In particular, light-induced redistribution of rod transducin from the outer segment to the inner segment and synaptic terminal (IS/ST) allows G?t1 and/or G?1?1 to modulate synaptic transmission from rods to rod bipolar cells (RBCs). Protein-protein interactions underlying this modulation are largely unknown. We discuss known interactors of transducin in the rod IS/ST compartment and potential pathways leading to the synaptic effects of light-dispersed G?t1 and G?1?1. Furthermore, we show that a prominent non-GPCR guanine nucleotide exchange factor (GEF) and a chaperone of G? subunits, resistance to inhibitors of cholinesterase 8A (Ric-8A) protein, is expressed throughout the retina including photoreceptor cells. Recent structures of Ric-8A alone and in complexes with G? subunits have illuminated the structural underpinnings of the Ric-8A activities. We generated a mouse model with conditional knockout of Ric-8A in rods in order to begin defining the functional roles of the protein in rod photoreceptors and the retina. Our analysis suggests that Ric-8A is not an obligate chaperone of G?t1. Further research is needed to investigate probable roles of Ric-8A as a GEF, trafficking chaperone, or a mediator of the synaptic effects of G?t1.
2020-01-01 | S-EPMC7591391 | BioStudies
Project description:G protein coupled receptors (GPCRs) can be activated by various extracellular stimuli, including hormones, peptides, odorants, neurotransmitters, nucleotides, or light. After activation, receptors interact with heterotrimeric G proteins and catalyze GDP release from the G? subunit, the rate limiting step in G protein activation, to form a high affinity nucleotide-free GPCR-G protein complex. In vivo, subsequent GTP binding reduces affinity of the G? protein for the activated receptor. In this study, we investigated the biochemical and structural characteristics of the prototypical GPCR, rhodopsin, and its signaling partner, transducin (G(t)), in bicelles to better understand the effects of membrane composition on high affinity complex formation, stability, and receptor mediated nucleotide release. Our results demonstrate that the high-affinity complex (rhodopsin-G(t)(empty)) forms more readily and has dramatically increased stability when rhodopsin is integrated into bicelles of a defined composition. We increased the half-life of functional complex to 1 week in the presence of negatively charged phospholipids. These data suggest that a membrane-like structure is an important contributor to the formation and stability of functional receptor-G protein complexes and can extend the range of studies that investigate properties of these complexes.
1000-01-01 | S-EPMC3119548 | BioStudies