A_2AR.

The protein-lipid interplay has been extensively studied for GPCRs with CHL [14]. CHL binding is reported to lead to conformational stabilisation of several GPCRs [66–68]. Addition of the more water soluble analogue CHS was previously shown to improve A_2AR stability and was crucial for purification and initial structure determination, which led to the identification of three different cholesterol binding sites [53,69]. Due to the well documented effects of CHL and CHS on GPCR stability, A_2AR serves as a control protein to test the ability of the lipid screen to identify stabilising lipids.

Ligand binding and high concentrations of NaCl were shown to further stabilise A_2AR [53]. Here, A_2AR was purified in the absence of CHS to test its effect on protein stability following our protocol for high-throughput lipid screening using the lipid screen. As expected, CHS stabilised the receptor with a ΔT_m of +10.8 ± 0.2°C (Fig 1C), in good agreement with previously determined values [69]. The next best hit was 16:0–18:1 PA with a ΔT_m of +5.8 ± 0.2°C (Fig 1C). So far, no functional or thermostabilising effects have been reported for PA’s on A_2AR. Besides CHL, several ordered lipid chains most likely originating from the LCP were found in A_2AR crystal structures, of which some seem to form specific interactions [69]. Previously, phospholipids have also been shown to occupy proposed cholesterol binding pockets [53]. More recent studies further imply a functional role of anionic phospholipids such as PIP₂ in receptor activation [15]. Phospholipids, particularly PA with a very small headgroup, might be able to bind to these lipid binding pockets and thereby stabilise the receptor in one particular conformation.

The addition of 50 μM of the A_2AR antagonist, ZM241385 (ZMA) served as an assay control, conferring an increase in T_m of +5.2 ± 0.5°C as expected (Fig 1C). This value is slightly lower than previously reported [53], which is probably due to the different assay buffer composition used (no CHS but high salt and theophylline present).

UapA.

Using the lipid screen, we identified previously unknown stabilising lipids for UapA. However, their effect was less pronounced compared to the CHS and 16:0–18:1 stabilisation of A_2AR. All PSs tested stabilised UapA, with POPS having the biggest effect (ΔT_m: +5.1 ± 1.4°C) (Fig 2C). Previous studies have indicated that UapA purifies in DDM in the presence of PI, PE and PC, with both PI and PE having roles in maintaining the functional dimeric state of the protein [12]. Whilst PI is too expensive to include in the screen and was thus not assessed here, PE had no effect on UapA. MD simulations showed that the PE binds less tightly than PI and the effects of both PI and PE could only be seen following delipidation and subsequent addition of the lipids [12]. Therefore, it is possible that PI and PE lipids that remain bound to the protein during isolation form tight interactions with the protein and additional PE has no significant effect. Interestingly, we did see a stabilising effect for PS. This is not a lipid identified from previous lipidomics analysis of purified UapA, suggesting it binds relatively weakly compared to PI and PE. PS does co-purify with the structurally related boron transporter from Saccharomyces cerevisiae (ScBOR1p) and has a role in dimer formation [50], strongly indicating that the precise effects of this lipid on UapA are worth further analysis. CHS also stabilises the protein with a ΔT_m of +4.2 ± 1.4°C (Fig 2C). UapA is a fungal protein and as such physiologically exposed to ergosterol, rather than the mammalian equivalent, CHL [70]. However, due to their structural similarity CHS likely substitutes for ergosterol in stabilising UapA [71]. Further analyses exploring the effects of CHL/ergosterol are warranted in the case of UapA. CL, a lipid that UapA does not encounter in the cell, markedly destabilises UapA (Fig 2C). PA’s also destabilise UapA although natively present in Aspergillus nidulans membranes [70]. Intriguingly, a combination of PE and PG also has a destabilising effect on the protein although DPPG alone stabilised the transporter with a ΔT_m of +3.9 ± 1.3°C.

The addition of the substrate, xanthine, did not stabilise UapA (Fig 2C). It is important to note that the construct we are using is a thermostabilised form of the protein (UapA-G411V_Δ1–11) trapped in the inward facing conformation [72]. Previous analysis indicated that the presence of xanthine stabilises this construct but only in protein that had been solubilised in the comparatively harsh detergent, nonyl-b-D-glucoside (NG). In the larger micelles of DDM, used to purify the protein for analysis here, the addition of xanthine appears to have minimal effects.

Tm-PPase.

Tm-PPase is a thermophilic protein and, as such, unfolds at much higher temperatures compared to the mesophilic UapA and A_2AR. To date, little is known about the effects of lipids on M-PPases. Hydrolytic activity measurements show increased activity of thermophilic M-PPases after relipidation with soybean lecithin [73]. However, soybean lecithin is not part of the lipid screen due to its short shelf life. Surprisingly, the analysis of the melting scan data reveals stabilising effects of lipids on Tm-PPase across almost all lipid classes represented in the screen, including anionic lipids POPG and POPS, with an overall average ΔT_m of +4.6 ± 0.4°C (Fig 3). Such broad stabilisation across different lipid classes is most likely conferred by non-specific interactions of protein with the lipidated detergent micelle rather than specific protein-lipid interactions (see Introduction). Indeed, Tm-PPase was purified in a rather harsh detergent, OGNG, used routinely for structural studies, due to the intrinsic stability of the protein [74]. The lipidation of detergent micelles likely reduces the micelle curvature, leading to increased protein stability [75]. The different effect on Tm-PPase stability observed within anionic lipids (e.g. 16:0–18:10PA vs. POPS) and PC’s (e.g. LMPC vs. DMPC) highlights the contribution of both lipid parts, the hydrophilic head group (changed in the former pair) and the hydrophobic lipid tail (changed in the latter pair) to their overall properties and effect on proteins (Fig 3C). Beside PA’s and LMPC, only MAG’s and CHS did not stabilise Tm-PPase solubilised in OGNG. Conversely, CHL addition slightly improved the thermostability (ΔT_m: +2.9 ± 0.6°C) (Fig 3C). Biophysical characterisation and atomistic simulations have previously shown that CHS is not an ideal mimic of CHL; even lesser so in its deprotonated form (pK_a,CHS: ~5.8), which is predominantly present in the Tm-PPase buffer (pH: 6.5), which may explain the differences observed between the two [76].

We also tested the effect of the non-hydrolysable inhibitor IDP, which is regularly used for the functional characterisation of M-PPases and in structural studies to lock the protein in a conformational state. IDP binding to Tm-PPase did not result in thermostabilisation suggesting that the IDP-bound structure is not energetically more favourable than the apo-structure (Fig 3C), even though IDP is known to protect Tm-PPase against protein degradation [77]. Biophysical data characterising the apo-structure of Tm-PPase and studies of Tm-PPase using a combination of lipids with IDP would yield insights into why IDP did not appear to be stabilising.

A_2AR.

We expressed and purified a stabilised human A_2AR construct (Δ317–412, 209–218 replaced with a thermostabilized apocytochrome b562 from Escherichia coli (BRIL)), which was successfully used for structural studies in the past [53,69]. Expression in a baculovirus expression system followed the manufacturer’s instructions for the Bac-to-Bac system (Invitrogen). Spodoptera frugiperda (Sf9) insect cells were infected at a cell density of 2 million cells/mL with P2 virus at a 1:2000 dilution. Infected cells were harvested after incubation for 60 hours at 27°C while shaking.

The purification is described in detail elsewhere [69]. In brief, Sf9 membranes were disrupted and harvested by repeated Dounce homogenisation and ultracentrifugation in hypotonic [10 mM MgCl₂, 20 mM KCl and 50 mM HEPES pH 7.5] and hypertonic [0.8 M NaCl, 50 mM HEPES pH 7.5] buffers supplemented with and 1x proteoloc protease inhibitor cocktail (Expedeon). The final membranes were resuspended in hypotonic buffer supplemented with 40% (v/v) glycerol and flash frozen in liquid nitrogen for storage at -80°C until used. Prior to solubilisation, the membranes were incubated with 2 mg/mL iodoacetamide, 4mM theophylline and 1x proteoloc protease inhibitor cocktail for 30 minutes at 4°C. The receptor was then extracted using 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM) for 4 hours at 4°C, followed by centrifugation at 150,000 xg for 1 hour at 4°C. The supernatant was incubated overnight with 1.5 mL packed HisPur cobalt resin (Fisher Scientific) per litre of culture in a final buffer containing 50 mM HEPES pH7.5, 0.8 M NaCl, 10% (v/v) glycerol 1 mM theophylline and 20 mM imidazole. The resin was washed with 10 column volumes (CV) wash buffer A [50 mM HEPES (pH 7.5), 0.8M NaCl, 10 mM MgCl₂, 10% (v/v) Glycerol, 25 mM imidazole, 0.1% (w/v) DDM, 8 mM ATP,1 mM Theophylline] followed by 4 CV wash buffer B [50 mM HEPES (pH 7.5), 0.8 M NaCl, 10% (v/v) glycerol, 50 mM imidazole, 0.05% (w/v) DDM, 1 mM theophylline]. The receptor was eluted in 9x 1 CV fractions elution buffer [25 mM HEPES pH7.5, 0.8 M NaCl, 10% (v/v) glycerol, 220 mM imidazole, 0.025 (w/v) DDM, 0.5 mM theophylline]. Elution fractions containing purified A_2AR were combined after SDS-PAGE analysis (S1 Fig). The purified protein sample was concentrated to 2 mg/mL with a 100 kDa molecular weight cut off (MWCO) Vivaspin centrifugal concentrator (GE Healthcare), and buffer exchanged to the final assay buffer [25 mM HEPES (pH 7.5), 0.8M NaCl, 1% (v/v) glycerol, 0.01% (w/v) DDM, 0.5 mM theophylline] using a PD10 desalting column (GE Healthcare). The final protein concentration was 1 mg/mL, determined using a detergent compatible protein assay (DC Protein assay, BioRad). Finally, the sample was flash frozen in liquid nitrogen and stored at -80°C.

UapA-G411V.

A modified version of UapA lacking the 11 N-terminal residues and containing a single point mutation UapA-G411V_Δ1–11 was expressed as a fusion protein with the C-terminal tobacco etch virus (TEV) cleavage site followed by GFP and 8xHis tag. UapA-G411V_Δ1–11 was transformed into the protease-deficient Saccharomyces cerevisiae strain FGY217 (MTAα, ura3-52, lys2Δ201, pep4Δ) described in detail elsewhere [80]. In brief, single colonies were inoculated in 10 mL minus-URA media [2 g/L amino acid mix w/o uracil, 6.7 g/L yeast nitrogen base w/o amino acids, 2% (w/v) glucose] in 50 mL aerated TubeSpin bioreactor tubes (TPP) and incubated at 30°C for 16 hours with 300 rpm shaking. The overnight cultures were diluted in 350 mL minus-URA media supplemented by 2% (w/v) glucose in a 1 L flask and incubated at 30°C for 24 hours, shaking at 300 rpm. The culture was then diluted with 1 L of minus-URA media supplemented with only 0.1% (w/v) glucose for induction at an OD₆₀₀ of 0.6. 22 hours after induction with 2% (w/v) galactose, cells were harvested at 4,000 xg for 10 minutes and the pellets were resuspended in ~6 mL of CRB buffer [50 mM Tris (pH 7.5), 1 mM EDTA, and 0.6 M sorbitol] per litre of cell culture. The cells were lysed using a Constant systems cell disruptor at 25, 30, 33 and 36 kpsi (5°C). Cell debris was removed by centrifugation at 10,000 xg for 10 minutes at 4°C. The membranes were harvested by ultracentrifugation at 100,000 xg for 2 hours at 4°C and resuspended in 6 mL MRB buffer [20 mM Tris (pH 7.5), 0.3 M sucrose, 0.1 mM CaCl₂] per litre of cell culture. Afterwards, membrane proteins were solubilised for 1 hour at 4°C in solubilisation buffer [1x PBS pH 7.5, 100 mM NaCl, 10% (v/v) glycerol, 1% (w/v) DDM, 1 mM xanthine, and one protease inhibitor tablet (Roche)] and insoluble matter was removed by centrifugation at 100,000 xg, for 45 minutes (4°C). The supernatant was supplemented with 10 mM imidazole (pH 7.5) and incubated for 2 hours at 4°C with 20 mL of nickel-nitrilotiacetic-acid (Ni-NTA) superflow resin (Quiagen) pre-washed in buffer A [1xPBS pH 7.5, 100 mM NaCl, 10 mM imidazole, 10% (v/v) glycerol, 0.03% (w/v) DDM, and 1 mM xanthine]. The mixture was loaded into an Econo-Pac gravity flow column (Bio-Rad) and washed with 6 CV of buffer A followed by 20 CV of Buffer A supplemented with 30 mM imidazole. The protein was eluted with 3 CV of buffer B [1x PBS pH 7.5, 150 mM NaCl, 10% glycerol, 310 mM imidazole, 1 mM xanthine, and 0.03% (w/v) DDM].

The protein concentration was estimated by measuring GFP fluorescence (Molecular Devices Spectramax M2) and TEV protease was added at a 1:1 ration of protease to protein. The sample was transferred to a dialysis membrane with a 12 kDa MWCO (Fisher Scientific) for 16 hours at 4°C for GFP-His tag cleavage. Following cleavage, the sample was supplemented with 12 mM imidazole, centrifuged for 10 minutes at 4,000 xg (4°C) and passed through a 0.22 μm filter to remove any precipitation. Afterwards, the sample was loaded on a 5 mL HisTrap column (GE Healthcare) pre-equilibrated with buffer C [20 mM Tris pH 7.5, 150 mM NaCl, 0.6 mM xanthine, and 0.03% (w/v) DDM] additionally containing 15 mM imidazole to remove TEV and cleavage product from the sample. The flow through was collected and concentrated to a volume of 0.5 mL in the Amicon 100 kDa MWCO concentrators (Merck) and injected into an equilibrated (buffer C) Superdex 200 10/300 gel filtration column (GE Healthcare). The sample was run at a flow rate of 0.35 mL/minute at 4°C. The quality of eluted fractions was checked by SDS-PAGE analysis (S1 Fig). The purest and most monodispersed fractions were pooled together, concentrated to 0.5 mg/mL using the Amicon 100 kDa MWCO concentrators (Merck) and flash frozen in liquid nitrogen for storage at -80°C until used.

Tm-PPase.

Protein expression and purification followed protocols described in detail elsewhere [73]. In brief, 6xHis-tagged Tm-PPase in the pRS1024 vector under the control of the PMA1 promoter was freshly transformed into the protease-deficient Saccharomyces cerevisiae strain BJ1991 (α prb1-1122 pep4-3 leu2 trp1 ura3-52 gal2) following a standard protocol for yeast transformation [80]. Colonies grown on yeast-peptone-dextrose (YPD) plates [1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose, 1.5% (w/v) agar, 100 μg/mL Carbenicillin] were transferred into 250 mL of synthetic Complete Dropout minus leucine (SCD-Leu) media [2.7% (w/v) yeast nitrogen base, 2% (w/v) glucose, 0.5 mM L-tryptophan, 0.4 mM L-histidine, 0.2 mM L-adenine, 0.2 mM L-uracil, 100 μg/mL Carbenicillin]. The culture was cultivated for 24 hours (30°C, 200 rpm) and used to inoculate 750 mL 1.5X YPD media for protein expression (8 hours, 30°C, 200 rpm). Afterwards, cells were harvested and washed twice in deionised water. The cell pellets were resuspended in 0.5 mL buffer [200 mM Tris pH 7.5, 40% (w/v) glycerol, 10 mM EDTA, 2 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonyl fluoride (PMSF)] per gram of dry cell mass for lysis using a bead beater and 0.5-mm glass beads. The bead beater chamber was topped up with 10 mM Tris pH 7.5, 10% (v/v) glycerol, 5 mM EDTA, and 1 mM DTT until completely full. Cells were lysed at 4°C by 12x 1-minute activations, interspaced by 1-minute cool-down periods on ice. Cell debris was removed (3,500 xg, 4°C, 15 minutes) and the supernatant was diluted with 10 mM Tris pH 7.5, 5 mM EDTA, and 1 mM DTT to a glycerol concentration of 20% (v/v). Membranes were harvested at 100,000 xg and 4°C for 1 hour and resuspended in 50 mM MES-NaOH pH 6.5, 20% (v/v) glycerol, 50 mM KCl, 5 mM MgCl₂, 1.33 mM DTT, 0.336 mM PMSF, and 2 μg/mL Pepstatin A. The total protein concentration of the extracted membranes was determined in a Bradford assay and diluted to 7.2 mg/mL. Diluted membranes were supplemented with 0.33 mM Na₂PP_i and mixed at a 3:1 ratio with 4x solubilisation buffer [50 mM MES-NaOH pH 6.5, 20% glycerol, and 5.34% (w/v) DDM]. Protein solubilisation followed the hot solve method [73] at 75°C for 1.5 hours. Denaturated proteins and insoluble matter were removed by centrifugation (4,000 xg, 15 minutes) and KCl was added to a final concentration of 0.3 M. 10 μL of Ni-NTA superflow resin was added per 1 mL of solubilised membrane sample. The mixture was loaded into an Econo-Pac gravity flow column after 1.5 hours at 40°C and 200 rpm shaking. The resin was washed with 2x CV of wash buffer [50 mM MES-NaOH pH 6.5, 20% (w/v) glycerol, 50 mM KCl, 5 mM MgCl₂, 20 mM imidazole, 0.05% (w/v) octyl glucose neopentyl glycol (OGNG), 1 mM DTT, 0.2 mM PMSF, and 2 μg/μL pepstatin A] followed by protein elution in 2x CV of 50 mM MES-NaOH, pH 6.5, 3.5% (v/v) glycerol, 50 mM KCl, 5 mM MgCl₂, 400 mM imidazole, 0.05% (w/v) OGNG, and 1 mM DTT. Imidazole was removed in a buffer exchange into 50 mM MES-NaOH, pH 6.5, 3.5% (v/v) glycerol, 50 mM KCl, 5 mM MgCl₂, 0.05% (w/v) OGNG) using a PD10 desalting column. The sample purity was checked by SDS-PAGE analysis (S1 Fig). Purified protein was concentrated to 1 mg/mL in 50 kDa MWCO Vivaspin 2 centrifugal concentrators, flash frozen in liquid nitrogen and stored at -80°C until used.