Methods

β₂AR expression and purification

Human β₂AR with N terminal epitope flag and truncated after residue 365 expressed in Sf9 insect cells using BestBac baculovirus system and incubated at 27˚C for 2 days.

Purified by ligand affinity chromatography and labelled with biotic-PEG₁₁-maleimide.

Any unlabelled receptor was blocked with 2 mM iodoacetamide and purified with alprenolol sepharose chromatography to isolate only functional receptors. Receptor was eluted and frozen in 20% glycerol.

Display and functional evaluation of Nb80 on yeast

Nb80 was cloned into the Aga-2 yeast display vector pYAL²⁰ and transformed into yeast.  Induced yeast which displayed the Nb80 on their cell wall were washed with PBE buffer and stained with biotinylated receptor bound to either BI167107 or carazolol.  The yeast was then washed with PBEM buffer and stained with Alexa-647-conjugated streptavidin for 15 minutes at 4˚C. 

Affinity maturation library of Nb80

 The affinity maturation library was prepared by assembly PCR with oglionucleotide primers, which contained degenerate codons at 15 known positions. These products were further amplified with primers containing 50 base pairs of homology to pYal. The pYal vector and mutagenic nanobody DNA were inserted into yeast cells. 

1^st round of selection: Yeast induced with SGCAA medium and resuspended in PBEM buffer, containing biotinylated β₂AR bound to BI167107. Then the cells were stained with Alexa-647-conjugated streptavidin. Yeast was then washed again in PBEM buffer and magnetically labelled with anti-Alexa-647 microbeads. This allowed yeast to be isolated with magnetic selection.

Rounds 2-6 were done using a similar method, but a few modifications were used. 

Negative selection of agonist-bound receptor was done to select for clones with a high preference to the active state of the receptor. Yeast cells were suspended in PBEM buffer containing biotinylated β₂AR bound to carazolol. Again they were labelled with Alexa-647 or PE-conjugated streptavidin, and then magnetically labelled. The magnetically labelled yeast was collected for subsequent positive selection. Cells binding the inactive form of the receptor were discarded.

Positive selection was then carried out as in round one, but using a decreasing concentration of BI167107 bound β₂AR was used. 

After round 6, the yeast were plated onto agar plates and colonies were picked out and cultured. The plasmids containing the nanobodies were isolated and sequenced.

Nanobodies were cloned into the pET26b vector which contained a C-terminal histidine tag, and transformed into the E. coli strain BL21(DE3). Expression was induced by IPTG overnight, and the periplasmic proteins were obtained by osmotic shock. The nanobodies were then purified using Ni²⁺ affinity chromatography due to the histidine tag. This tag was then removed. 

Purification and crystallisation of β₂AR -Nb6B9 complexes

Human β₂AR was fused to a T4 lysozyme and expressed in Sf9 insect cells. Purification was carried out as before. The receptor was then washed with the low affinity ligand atenolol to fully displace the alprenolol. The receptor was then incubated with HBI or BI167107 ligands, and an excess of Nb6B9 was added. The samples were then concentrated, and the β₂AR -Nb6B9 complex isolated. For the adrenaline-bound receptor complex, adrenaline was added to a sample of β₂AR and an excess of Nb6B9 was added.

After purification samples were concentrated to A_{280 nm} =55, and aliquoted into thin-wall PCR tubes. These were flash-frozen. For crystallisation these samples were thawed and reconstituted into lipidic cubic phase with lipid (acylglycerol and cholesterol). In the case of the adrenaline-receptor complex, fresh adrenaline was added again before reconstitution due to its instability. The samples were placed on glass sandwich plates and crystals were grown using 30 nl protein/lipid drops with 600 nl overlay.

Crystallographic data

X-ray diffraction data was collected at Advanced Photon Source GM/CA beamlines 23ID-B and 23ID-D. A 10 μm beam was used with 2s exposure, 0.6˚ oscillation and no beam attenuation.