Abstract
G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal transduction and to facilitate optimal drug design. However, due to their inherent instability, crystallisation of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and β-arrestins, has proved challenging. Here, we describe the design of a minimal G protein, mini-Gs, which is composed solely of the GTPase domain from the adenylate cyclase stimulating G protein Gs. Mini-Gs is a small, soluble protein, which efficiently couples GPCRs in the absence of Gβγ subunits. We engineered mini-Gs, using rational design mutagenesis, to form a stable complex with detergent-solubilised β1-adrenergic receptor (β1AR). Mini G proteins induce similar pharmacological and structural changes in GPCRs as heterotrimeric G proteins, but eliminate many of the problems associated with crystallisation of these complexes, specifically their large size, conformational dynamics and instability in detergent. They are therefore novel tools, which will facilitate the biochemical and structural characterisation of GPCRs in their active conformation.
Highlights
G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling, through heterotrimeric G proteins and β-arrestins, in response to extracellular stimuli, such as hormones and neurotransmitters (Rosenbaum et al, 2009)
We hypothesised that this domain would be sufficient to stabilise GPCRs in their fully active state, i.e. the conformation adopted by β2-adrenergic receptor (β2AR) in the β2AR–Gs complex (Rasmussen et al, 2011b), and it was used as the starting point to engineer a minimal G protein
The β2AR–Gs complex provided the first insight into the organisation of the native GPCR–G protein interface, which is something that other binding proteins cannot recreate, but frustratingly, complexes involving heterotrimeric Gs are the most difficult to crystallise, due to their large size and dynamic nature
Summary
G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling, through heterotrimeric G proteins and β-arrestins, in response to extracellular stimuli, such as hormones and neurotransmitters (Rosenbaum et al, 2009). GPCRs adopt different conformational states in response to binding different classes of ligand and coupling to cytoplasmic signalling proteins. Structure determination of GPCRs in all activation states is important to decipher the molecular mechanisms of signal transduction, and to facilitate optimal drug design. Heterotrimeric G proteins are composed of α, β and γ subunits. Gα consists of a GTPase domain (GαGTPase), which is analogous to members of the small GTPase superfamily of proteins, and an αhelical domain (GαAH), which is unique to heterotrimeric G proteins (Sprang, 1997). GDP-bound state, Gα binds Gβγ, forming a heterotrimer with low basal nucleotide exchange activity (Higashijima et al, 1987). The trimer is anchored to the cell membrane, through lipid modifications of both Gα and Gγ
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