Structure of activated Gs

Figure 1 - Structure of the different subunits of Gs with Nb35
bound in the interface between Gβ and GαsRas

The experiment also focused on the specific structure of the Gs protein and its subunits in its active state. The G-protein consists of three subunits: Gαs, Gβs and Gγs. The Gαs also consists of two domains (Figure 2):

• GαsRas - Ras-like GTPase domain which interacts with the β₂AR
• GαsAH - α-helical domain



Figure 2 - Structure of the Gαs domains interacting with the
receptor (Gβγ domains hidden).



The β₂AR-Gs complex was compared with that of the GTP-bound active state by the use of GTPγS, an analogue of GTP, to observe any conformational changes. The active Gαs–GTPγS structure showed a displacement of the GαsAH domain relative to the GαsRas by a rotation of approximately 127° at the point of interaction between the two domains (Figure 3).

Figure 3 - Comparison of the Gαs domains in the Gαs–GTPγS 
and the β₂AR-Gs complex (Soren G. F. Rasmussen et al., 2011).



A study by Van Eps, N. et al. (2011), revealed that the binding pocket for the nucleotide (GTP) is located at the interface between GαsAH and GαsRas in which the interaction between the two are stabilised by the binding of a guanine nucleotide.

This shows the importance of the interaction between GαsAH and GαsRas domains for the binding of GTP to subsequently allow the G-protein to become activated and reduce the interaction between Gα and Gβγ to allow Gα to activate downstream signalling proteins. [More details]

The GαsRas and GαsAH domains can together form a functional GTPase when they are expressed together as separate proteins which may explain the displacement of GαsAH.

The interaction between the GPCR and the GTP-binding pocket involves the N- and C-terminal helices of Gαs. The most significant conformational changes in the GαsRas are 

• the displacement of the α5-helix towards the receptor by 6Å and rotation as the C-terminal projects towards the TM core of the receptor.* 
• Another conformational change in the GTP-activated G-protein is the β6-α5 loop which is displaced away from the GTP-binding pocket.

*The α5-helix has been shown to be important in the interaction between the β₂AR and the G-protein. 

Although the β₂AR-Gs structure has been successfully compared with that of the active Gαs–GTPγS, the structure of the GDP-bound Gs heterotrimer has not been determined yet. [More details]

------------------------------------------------------------------------------------------------------------

Van Eps, N. et al. (2011):
 The study used double electron-electron resonance (DEER) spectroscopy demonstrated the change in the interaction between the two domains induced by the G-protein’s interaction with an activated receptor; rhodopsin in this case. The formation of the complex of Gi interacting with light-activated rhodopsin demonstrated its induction in the opening of the interface between the two domains to allow the dissociation of GDP from the binding pocket and allow the exchange for GTP.[1]

Unrevealed structure of the GDP-bound Gs:
This means that the structures of the active and inactive Gs have not been able to be compared with its association with the β₂AR. However, study with GDP-bound Gi in complex with β₂AR has shown the lack of significant conformational changes between the GαsRas and Gβ subunit. This was carried out by the use of Nb35 which means that possible effects of Nb35 on the orientation of the GαsRas-Gβγ interaction should be taken into account in the crystal structure.

---

Reference:
[1] Van Eps, N. et al. Interaction of a G protein with an activated receptor opens the interdomain interface in the a subunit. Proc. Natl Acad. Sci. USA 108, 9420–9424 (2011).