Structure of the active-state β2AR

Figure 1 - Comparison of the active and inactive structures of β₂AR,
with the agonist represented in green.



 

Active vs Inactive

The experiment looked at the structure of the ligand-bound active β₂AR structure and determined the conformational changes which occur to understand the proteins' ternary structure in relation to its functional properties. Comparison of the structures between the active agonist-bound receptor in the β₂AR-Gs complex (red) and the inactive carazolol-bound β₂AR (blue) was used to work out the conformational changes in the protein (Figure 1&2):



      - 14Å outward movement of TM6
- Outward movement and extension of the cytoplasmic end of the TM5 helix by 7 residues.
- A stretch of 23 amino acids in the third intracellular loop (ICL3) is disordered.
- The ICL2 forms an extended loop in the inactive carazolol-bound β₂AR structure and an α-helix in the β₂AR-Gs complex (but this may not be unique to the active state as it is also observed in the inactive homologous avian β₁AR).

Figure 2 - Cytoplasmic view of the active and inactive structures of β₂AR

Stabilisation

The electron density maps for the extracellular side of the receptor were of low quality due to its flexibility and the lack of stabilising interactions for packing, such as those by adjacent receptors. This problem was resolved by indirectly tethering the protein to T4 lysozyme by amino-terminal fusion (Crystallisation method).



The structure of the T4L-β₂AR-Gs complex was compared to the active-state structure of β₂AR which was stabilised by Nb80, a G protein mimetic nanobody, which allows more efficient packing interactions with adjacent receptor molecules to increase the resolution of the electron density maps (good for a 3.5Å).,[i]

The β₂AR-Gs and β₂AR-Nb80 structures have high resemblance with only significant differences at the cytoplasmic ends of the TM helices 5 and 6 where they interact with different G-proteins but also have highly conserved sequences (E/DRY and NPxxY) which have been shown to be important in the activation or maintaining the receptor in the inactive state.

Therefore, both structures have shown similarity in the position of residues apart from Arg131 which interacts with different proteins in the two complexes:

- Arg131 of β₂AR-Gs packs against Tyr391 of Gαs
- Arg131 of β₂AR-Nb80 interacts with Nb80

Overall, both structures show similarity in both function and structure, with high affinity for the agonist isoproterenol, demonstrating high structural homology around the ligand binding pocket.

 

 

Interaction with GαsRas

The active state of β₂AR is stabilised by its interaction with the GαsRas subunit which is formed by the ICL2, TM5 and TM6 of the β₂AR with the α5-helix, αN-β1 junction, the top of the β3-strand and the α4-helix of GαsRas. The G protein coupling, however, does not have evidence of any consensus sequence in the β₂AR for specificity which is likely when taking into account their ability to bind to other G proteins such as Gi. However, there are a few important residues in the interaction which include Phe 139 on the ICL2 helix that sits in the hydrophobic pocket formed by Gαs.

The β₂AR does not interact with the Gβγ directly, although Gs acts as a heterotrimer for efficient interaction with a GPCR. However, Gβ still has an important role in coupling by stabilising the N-terminal α-helix of Gαs. It is possible, based on findings of other models on GPCR dimers, that one of the protomers may interact with the Gα subunit whilst the other interacts with the Gβγ subunits.

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Reference:

[i] Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the b2

adrenoceptor. Nature 469, 175–180 (2011).