Crystallisation of the complex

The β₂AR-Gs complex is made up of a β2 Adrenergic Receptor bound to a Gs protein. In this sections we will look at how this receptor and G protein are produced and how the complex between them is formed then crystallised. 

β₂AR

T4 lysozyme was fused to the N-terminus of the β2AR in order to increase the stability of the receptor (see figure 1). A baculovirus construct (T4-LB2AR) with the T4 lysozyme bound to the N-terminus of the receptor was produced and used to infect Sf9 insect cells (from the Fall Armyworm) and solubilised in dodecylmaltoside (DDM).  

• The T4 lysozyme is fused to the N-terminus of the β2AR, but packs at the N-terminus of the Gβ subunit of the complex, the C-terminus of Gγ of another complex, and Gα subunit of another complex. As T4 lysozyme packs to a number of different complexes, it further strengthens the crystal contacts, hence improving the stability of the complex.

Figure 1: Shows the fusion of the T4 lysozyme to the N-terminus of the β2AR. The link between the two molecules is too flexible to be seen by x-ray crystallography and therefore the molecules appear as two separate units.

To generate a functional form of the receptor, two types of chromatography were carried out: firstly, M1 flag  affinity chromatography follwed by alprenol-sepharose chromatography, which was used to identify the functionally active receptor. A second M1 flag affinity chromatography was carried out to exchange the receptor bound to alprenol for a receptor bound to a high affinity agonist (BI167107). The receptor bound agonist was then eluted, treated with lambda phosphatase and concentrated.

Gs protein

Gs has three 3 subunits α , β and γ, each of which are coded for by different genes. In order to produce the Gs protein, each of these specific genes was placed into an individual virus which were then used to infect insect cells. The cultures were incubated for 40-48 hours before being harvested. The cells then underwent many centrifugation and purification steps. After the addition of further chemicals, which are listed in Rasmussen's paper, the protein was concentrated using a Millipore.

Forming a complex to be crystallised:

Now that we have identified how the receptor and Gs protein are produced, we need to combine the two to form a stable complex before the crystal can be produced.

A stable form of the complex was produced by adding 100 mM of GDP-Gs to an excess of agonist bound β₂AR in DDM solution. GDP disrupts interactions between β₂AR and the Gs protein; meaning it must be removed. Apyrase (a non selective purine phosphatase) was added to hydrolyse the GDP, thereby removing it from the complex.

Next, size exclusion and antibody affinity chromatography were used to purify the complex, which was then exchanged into NG-310: Anatrace (a maltose neopentyl glycol detergent). This exchange allowed the complex to be stored at room temperature for 24 hours without degradation.   

In order to aid the crystallisation, a nanobody (Nb35) was bound to the complex (forming T4L-β₂AR –Gs-Nb35 complex). The nanobody's function was to prevent the GTPγS from dissociating from the complex.

• Nb35 was produced by expression of the nanobody in the periplasm of E-coli WK6 strain, then extracted by nickel affinity chromatography followed by ion exchange.
• It interacts with the CDR1 region of the Gβ and the long CDR3 loop of the Gβ and Gα subunits, which explains why the NB35 can be found heavily packed at the interface of these two subunits.

Crystallisation of the β₂AR-Gs complex

The new T4L-β₂AR-Gs-Nb35 complex (Figure 1 in introduction) was mixed with a lipid designed to accomodate membrane proteins with large hydrophilic surfaces (MAG7.7) and a detergent (MNG-3). The function of MNG-3 was to maintain the structure of the complex and help generate crystals of a higher quality and size.

The protein lipid mixture was robotically dispensed into glass sandwich plates and a number of screenings were carried out to identify the exact conditions required to generate optimum crystals for data collection. 

The obtained crystals reached their full size of 250 microns in 4 days and they diffracted to 2.9 Å.

Figure 2: Lattice packing of the complex. Layers of alternating G protein and receptor can be seen within the crystal. (PDB ID: 3SN6)

The crystalline structure of the complex

Each asymmetric unit contained a single β₂AR-Gs complex.

There are alternating layers of lipid and solution (figure 2), but the crystal contacts only occur in the soluble layers. This results in the receptor molecules being widely separated out from one another in the membrane and being suspended by the Gs protein layers.