Trends in Pharmacological Sciences
ReviewFeature ReviewGPCR activation: a mutagenic spotlight on crystal structures
Section snippets
GPCR structure–function relationships
The seven-transmembrane (TM)-domain GPCRs are the largest superfamily of signalling molecules in the human genome. They provide a higher proportion of the actual and prospective targets for drugs used in the clinic (and, notoriously, for illicit recreational purposes) than any other protein family [1].
Our understanding of the structure–function relationships of GPCRs has made several spectacular advances. The greatest was arguably the cloning and sequencing of the classical post-synaptic
A conserved G-protein-binding microdomain
In addition to the ACh counter-ion D3.32, Ala-scanning mutagenesis of the TM domain of the M1 mAChR identified five amino acids, D2.50, I3.46, R3.50, N7.49, and Y7.53 [designated by the Ballesteros–Weinstein (BW) nomenclature; in extracellular loop (ECL) 2, residues are identified relative to the disulfide-bonded Cys], whose substitution reduced the signalling efficacy of ACh by more than 100-fold (Table 1). Their importance is reflected in the very high conservation of their homologues in the
Functional domains in the receptor conformational change
The very highly conserved residue D2.50 is at the root of the G-protein-binding microdomain that forms in the activated state. This suggested the hypothesis that TM2 might be regarded as the pivot of the activating conformational change. Pursuing this idea, superposition of TM2 between residues 2.38 and 2.65, spanning D2.50, in the inactive- [21] and active-state [35] structures of the β2AR yielded surprisingly precise superposition of the indole rings of Trp residues in different TM domains of
The receptor domains are functionalised by specific structural motifs
In this section, we show how specific structural motifs mediate the functions of the domains defined above. Of these motifs, two have a stabilising role, six are primarily concerned with regulating signal transduction, and three are specialisations to bind ligands. Some are generated by local sequence elements, whereas others involve inter-domain interactions.
Agonist activation: integration of the components of the conformational change
Activation requires movement of the cytoplasmic terminus of TM3 bearing the G protein contact residue R3.50 towards TM7, and partial unwinding of the C-terminal helix of TM7 to release Y7.53 to stabilise R3.50 and actuate G protein binding. These rearrangements require rupture of the steric clamp that inhibits the outward movement and reorientation of the cytoplasmic end of TM6. The core interactions in the TM6 clamp are highly conserved in all GPCR crystal structures determined so far. It is
Concluding remarks
Comprehensive mutagenesis studies have successfully spotlighted the functionally important residues in GPCR sequences, focusing our attention on particular elements of the crystallographic structures. These complementary approaches, informed by increasingly powerful molecular dynamics calculations, are converging to provide a view of GPCR activation that is both modular and dynamic. The activation of GPCRs may best be modelled by ligand-biased stochastic transitions between different ensembles
Acknowledgements
This work was supported by the Medical Research Council UK, grant-in-aid number MC_U117532184. Structures were visualised and aligned, intramolecular cavities were mapped, and figures were created with Pymol (Schrodinger Inc.). I am grateful to Dr Phil Walker, Division of Molecular Structure, NIMR, for computational assistance.
Glossary
- Agonist
- ligand that binds to a receptor and stabilises the active state.
- Ballesteros–Weinstein (BW) nomenclature
- positions of amino acids (single or triple letter code) in TM domain X are indicated relative to the most conserved residue, designated X.50.
- First shell
- immediate neighbours surrounding a particular set of amino acids
- Gq
- Gq type heterotrimeric (αβγ subunit) G protein.
- Gt
- transducin type heterotrimeric (αβγ subunit) G protein.
- Gs
- Gs type heterotrimeric (αβγ subunit) G protein.
- Indel
- mutation in
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