Structure of active state β2AR

Active State vs Inactive State

Through determining the structure of β₂AR-Gs, the mechanism of signal transduction across plasma membrane and various functional properties can be understood. Structure of the active agonist-bound receptor in the β₂AR-Gs complex and the inactive carazolol-bound β₂AR were compared in order to determine the conformational changes in GPCRs.

Differences:

• 14Å outward movement at cytoplasmic end of transmembrane segment 6 (TM6).
•  Outward movement and extension of the cytoplasmic end of the TM5 helix by 7 residues.
•  A disordered stretch of 26 amino acids in the third intracellular loop (ICL3).
• The ICL2 forms an extended loop in the inactive β₂AR structure and an α-helix in the active β₂AR-Gs complex

Figure 3a. Side view of overall structures of inactive and active states.

Figure 3b. Cytoplasmic view of overall structures of inactive and active states

Instability of Extracellular Receptor

Due to the lack of packing interactions with adjacent receptors, the extracellular half of the GPCR is more flexible and dynamic. The protein is indirectly tethered by the amino-terminal fusion to T4 lysozyme. This results in structural heterogeneity in the extracellular region, limiting the quality of the electron density map. Alternatively, β₂AR-Nb80 crystal was studied. G protein mimetic nanobody (Nb80) helps stabilize β₂AR by intensifying the packing interactions with adjacent receptors, which enhances the resolution of the map.

 β₂AR-Gs vs β₂AR-Nb80 

The two structures have huge similarity. Highly conserved sequences (E/DRY and NPxxY), have shown to be imperative for activation and maintaining the receptor in the inactive state. The only significant difference within the residues is Arg131 as they interact with specific proteins. β₂AR-Gs structure Arg131 packs against Tyr391 of Gαs while Arg131 of β₂AR-Nb80 interacts with Nb80. Both complexes show the same high affinity for the agonist isoproterenol, in evidence of the high structural homology around ligand binding pocket.

Interaction of β₂AR with GαsRas

The active state of β₂AR is stabilised by its interaction with the interface of GαsRas. This interface is formed by 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. Some of these sequences play a part in G protein coupling. However, as there is no evidence of consensus sequence in β₂AR for specificity, structural basis of G protein coupling is likely to be determined by several secondary and tertiary structures.

It is to our surprise that β₂AR has no direct interactions with Gβγ subunits. Nevertheless, Gβ plays a big part in G coupling by stabilising the N-terminal α-helix of Gαs. Studies also suggest that one of the promoters interact largely with the Gα subunit whilst the other interacts with the Gβγ subunits.