Isolation and structure–function characterization of a signaling-active rhodopsin–G protein complex

Abstract

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 (G T ). This results in the dissociation of G T into its component ␣ T -GTP and ␤ 1 ␥ 1 subunit complex. Structural information for the Rho*-G T 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 (G T *) 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 G T *. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the ␤ 2 -adrenergic receptor-G S complex, including a flexible ␣ T * helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a highresolution structure of this important signaling complex.G protein-coupled receptors (GPCRs), 3 the largest family of transmembrane proteins, are the targets for nearly 50% of all pharmaceutical drugs (1). These receptors modulate cellular responses to a vast array of extracellular signals through the activation of heterotrimeric G proteins, with the active state being defined as the complex that forms between the agonistbound (or light-stimulated) GPCR and nucleotide-free G protein (2). Attempts to obtain structural information for GPCRs, and especially for signaling-active GPCR-G protein complexes, have garnered a great deal of interest, both as a means to better understand the underlying mechanisms by which this important family of receptors mediates a wide range of biological outcomes and as a critical step in the design of more selective and effective drug treatments. Recent technological advancements, including novel protein engineering (3), in meso crystallization (4), and micro-focus beamlines at synchrotron facilities (5), have ushered in significant progress in the determination of high-resolution GPCR structures. However, only a few of those structures are of activated GPCRs, and thus far, only two GPCR-G protein complex structures have been solved, namely that of the ␤ 2 -adrenergic receptor-G S protein complex (6) and the calcitonin receptor-G S protein complex (7). Furthermore, virtually all of these structures, with the exception of rhodopsin, have been obtained with G...

Structural insights into emergent signaling modes of G protein–coupled receptors

“…Notably, this study revealed the putative role of the Gb 1 and the a-helical domain (aHD) of the Ga subunit in the G protein activation process. Whereas aHD is highly flexible (107)(108)(109) and therefore it remains unresolved in other GPCR/G protein cryo-EM structures, Gao et al (108) solved the structures of three Rho/G T complex conformers (at 4.5 Å) that differed in degrees of aHD opening (66°, 68°, and 91°compared with GDP-bound G T , but none as extensively displaced (132°) as aHD in the b 2 AR/Gs crystal structure). Most notably, in all of the three structures, aHD appears to make polar contacts with the Gb 1 subunit, which were found to be crucial to support optimal GDP!GTP exchange rate.…”

Shining a light on GPCR complexes

“…The new strategies and analysis from Gao et al (6) are different from previous X-ray and cryoEM studies in several ways. First, the authors isolated native bovine rhodopsin and the obligate heterodimer ␤1␥1 complex from photoreceptors, which they paired with a chimeric G␣ T subunit.…”

“…The structural model by Gao et al (6) provides new information relevant to two outstanding questions: One concerns the large movement of the G␣ helical domain, thought to be necessary for release of GDP from the deeply buried active site. This movement is one of the unique features of the GPCR-G protein complex that was first revealed in spectroscopic experiments of G␣ i activation by rhodopsin (7) and then visualized upon crystallization of ␤ 2 AR-G S (1).…”

A Rhodopsin-Like Gene May Be Associated With the Light-Sensitivity of Adult Pacific Oyster Crassostrea gigas

“…Rhodopsin is a major component of rod photoreceptors, which are photosensitive, even under extremely weak light conditions (Hargrave and McDowell, 1992 ). The light-activated rhodopsin is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (Gao et al, 2017 ).…”