Article pubs.acs.org/crystal
Effect of Lipidic Cubic Phase Structure on Functionality of the Dopamine 2L Receptor: Implications for in Meso Crystallization Connie Darmanin,*,†,‡ Sampa Sarkar,§ Laura Castelli,‡ and Charlotte E. Conn*,§,∥ †
ARC Centre of Advanced Molecular Imaging, Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia ‡ CSIRO Manufacturing, 385 Royal Parade, Parkville, Victoria 3052, Australia § RMIT, School of Science, College of Science, Engineering and Health, GPO Box 2476, Melbourne, Victoria 3001, Australia ∥ CSIRO Manufacturing, Private Bag 10, Clayton South MDC, Victoria 3169, Australia S Supporting Information *
ABSTRACT: The success of the lipidic cubic phase for crystallization, particularly of integral membrane proteins, is increasing. In the past two years, more than 25% of membrane protein structures have been solved within the biomimetic environment of the lipidic cubic phase. However, the relationship between the lipid matrix and crystal growth still remains a mystery. Herein we show that the bilayer structure of the lipidic cubic phase is crucial to retention of the functionality of the dopamine D2 long receptor. Destruction of the cubic architecture at higher protein concentrations is associated with a significant drop in the amount of functional receptor. This has profound implications for in meso crystallization and suggests that preliminary experiments to determine the maximum protein loading within the lipidic cubic phase must be carried out prior to in meso crystallization experiments.
1. INTRODUCTION G protein-coupled receptors (GPCRs), a group of integral membrane proteins associated with numerous diseases,1−4 are the target for more than 70% of prescription pharmaceutics currently available.1 The dopamine receptors belong to the GPCR super family of seven transmembrane domains. They have been implicated in a number of neurological diseases including Parkinson’s disease, schizophrenia, and Alzheimer’s disease.3−5 The dopamine 2 (D2) receptor is one of the five receptor subtypes (D1−D5).6 Within this subtype there are two alternative spliced isoforms, D2 short (D2S) and D2 long (D2L),7 which differ by an insertion of 29 amino acids in the third intracellular loop (ICL3), the region that interacts with G proteins.8 Activation of D2 dopamine receptors results in inhibition of adenylyl cyclase and a number of cell-type specific responses6,9 and is thought to be the target for many antipsychotic drugs, known to be dopamine antagonists.10,11 The significant level of adverse side-effects currently associated with antipsychotics directly results from nonspecific binding of the drug to all five subtypes of the dopamine receptors.12,13 Structure-based drug design may alleviate this problem by isolating pockets of these receptors which are not highly conserved and can be targeted to confer drug specificity between the subtypes. However, GPCRs, in general, are difficult to crystallize; it is only in the last 10 years, with the application of in meso crystallization technology, that a number of GPCR structures have been solved2,14−21 including the dopamine D3 receptor.22 © 2016 American Chemical Society
One issue with lipidic cubic phase (LCP) crystallization of many GPCRs is that it typically involves altering the ICL3 loop region by inserting a modified T4-lysozyme protein to induce crystal contacts.16,22,23 Unlike other GPCRs crystallized, this is an issue for the D2 receptor as the ICL3 of the D2 receptors is important for distinguishing the function between the two isoforms, D2L and D2S. Therefore, crystallization of the native, unmodified, receptor is essential for a complete understanding of its biological function. In meso crystallization uses the inverse bicontinuous cubic lipidic mesophases (otherwise known as the LCP) as a matrix for the growth of protein crystals.24 The unique amphiphilic nature of the LCP allows for the accommodation of hydrophobic, hydrophilic, or amphiphilic molecules, while the bicontinuous nature of both the lipid bilayer and the water channel networks is essential for crystallization, along with other applications such as controlled drug delivery.25,26 To date, the lipid monoolein and similar structural analogues have been used fairly ubiquitously for in meso crystallization. However, the mechanism of in meso crystallization, and the effect of the lipid mesophase on crystal growth, remain poorly understood; as of February 2016, of 595 unique entries deposited into the MP databank only 36 unique G-protein coupled receptor structures have been solved.27 Partitioning of Received: April 15, 2016 Revised: August 8, 2016 Published: August 16, 2016 5014
DOI: 10.1021/acs.cgd.6b00576 Cryst. Growth Des. 2016, 16, 5014−5022
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the detergent (from detergent solubilized protein), along with other crystallization additives into the bulk LCP, is believed to promote formation of a localized lamellar structure around the protein crystal, acting as a conduit between the lipidic cubic phase and the growing crystal.28 This lamellar phase has been directly observed for crystals of bR29 and the transmembrane complex of DAP1230 using SAXS; a number of studies have provided additional indirect evidence for this phase using freeze-fracture electron microscopy (EM)31 and atomic force microscopy (AFM).32 Several factors are known to affect soluble protein crystallization including temperature, pH, protein concentration, and the addition of crystallant. For in meso crystallization, the evolving mesostructure of the underlying lipid matrix must also impact crystal growth and adds an additional layer of complexity to this technique. The choice of lipid is believed to be crucial to crystal growth, and monoolein, which is not an endogenous lipid and has limited biological relevance, may not be the right choice for all proteins. However, the effect of different LCP systems on crystallization remains a significantly underexplored area of this growing field. Previous research has shown that incorporation of a protein or peptide has a significant effect on the structure of the lipid mesophase, potentially impacting the outcome of crystallization trials. Recently, protein loading of the brain-derived neurotrophic factor (BDNF) into the cubic phase was estimated using time-resolved SAXS (TR-SAXS).33−42 Herein we investigate the relationship between the lipid mesophase structure and the functionality of D2L receptor which is embedded within it. Functionality was determined via radiolabeled ligand-binding assays, which are routinely used for GPCRs to assess the amount of ligand bound to a particular receptor and indicate whether the protein has retained a protein conformation that preserves the ligand binding region. Retention of protein functionality is essential during crystal growth to ensure that the structure determined is that of the active protein conformation. We demonstrate that the amount of functional D2L receptor within the lipidic cubic phase (as determined by spiperone binding data) varies significantly with the protein concentration for three different lipids: monoolein (MO), phytantriol (PT), and phytanoyl monoethanomide (PE), Figure 1. These lipids vary in chain architecture: MO has
Table 1. Typical Bilayer Thickness and Water Channel Diameter for Monoolein, Phytantriol, and Phytanoyl Monoethanolamide at 20 °Ca lipid (chain length) monoolein (C18) phytantriol (C14) phytanoyl monoethanolmide (C16)
bilayer thickness (Å)
water channel diameter (Å)
fold increased in functional D2L receptorb
33.2 28.2 26.0
46.2 23.4 23.2
586 182 63
a
The increase in receptor functionality in each of these systems is provided at 0.5 mg/mL D2L concentration with respect to the soluble D2L sample at the same concentration. The water contents used in the calculations were the excess water points: 48%,43 28%,44 and 30%45 w/ w, respectively. The densities used in the calculations were 0.942 g cm−3, 0.940 g cm−3, and 0.940 g cm−3. bAt 0.5 mg/mL D2L concentration with respect to the soluble D2L sample at the same concentration (Figure 2B).
protein incorporation. Preincubation of the lipid within the lipidic cubic phase is also shown to be important; ideal preincubation times varied between 1 and 4 h for the different LCPs. We discuss how the structural evolution of the cubic phase following protein incorporation affects receptor functionality, and the possible impact of this on crystallization trials, including why in meso crystallization is notorious for producing small crystals.
2. EXPERIMENTAL SECTION 2.1. Lipids. Phytanoyl monoethanolamide (PE) was synthesized as previously described.45 Monoolein (M7765) was purchased from Sigma-Aldrich. 3,7,11,15-Tetramethyl-1,2,3-hexadecanetriol (phytantriol) was provided by DSM Nutritional Products, Germany. 2.2. Dopamine 2 Long (D2L) Receptor Expression and Purification. Full-length human D2L receptors were expressed in Sf 21 cells and purified using nickel affinity chromatography as described in Darmanin et al. 2012.46 The purity of the proteins was checked, and the protein concentration was determined using standard biochemical assays. 2.3. Radioligand Binding Assay. Functional studies on the D2L were carried out via a well-established radioligand binding assay using the [3H]-spiperone antagonist. The complete method has been published in Darmanin et al. 2012.46 1 mg of lipid (5 μL of lipid solution at 200 mg/mL in ethanol) was added to each well of a Millipore MultiScreen HTS 96-well filter plate. Solvent was evaporated initially for 12 h in a vacuum oven (40 °C; 0.2 MPa) and then in a fume hood for a period of at least 1 day. The purified receptor (0.69 μL in aqueous solution) was dispensed on top of the dried lipid film to produce a lipid mesophase with protein incorporated, unless explicitly stated otherwise, in the ratio 60:40 of lipid to protein. The protein− lipid mesophase was incubated for a period of 1, 2.5, or 4 h before addition an appropriate volume of TMN buffer (50 mM Tris, 10 mM MgCl2, 100 mM NaCl, pH 7.6, total assay volume was 50 μl). Unlabeled spiperone (2.1 μM) was used to determine nonspecific spiperone binding. Tritiated ligand binding detection was carried out in a Wallac MicroBeta TriLux 1450LSC and Luminescence Counter (PerkinElmer) in the presence of Ultima Gold (PerkinElmer) liquid scintillant (50 μL). The binding data were analyzed using GRAPHPAD PRISM4 (GRAPHPAD Software, Inc., San Diego, CA, USA), and the ligand affinities were determined based on Swillens47 global fitting procedure. Note that the spiperone binding assay is representative of ligand binding and not the full functionality of the receptor with respect to the G protein coupling. Therefore, when we state an increase in functional binding we relate this to the fact that the receptor is folded
Figure 1. Chemical structure of (a) monoolein, (b) phytantriol, and (c) phytanoyl monoethanolamide.
a singly unsaturated C18 chain, while PT and PE both contained a branched, isoprenoid-type C16 hydrocarbon chain. They adopt LCPs with significantly different bilayer thickness and water channel diameter as detailed in Table 1. We show that protein functionality is strongly associated with corresponding structural changes to the lipidic cubic phase following 5015
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Figure 2. The effect of the change in concentration of the dopamine 2 long (D2L) receptor on three different lipid systems. The specific binding of spiperone to D2L is shown in three different lipid systems; monoolein (MO, purple), phytantriol (PT, black), and phytanoyl monoethanolamide (PE, green). (a) Spiperone binding shown in the three different lipid systems at a range of concentrations from 0.5 mg/mL to 7.8 mg/mL. (b) The percentage of functional receptor in the assay volume is shown for each concentration. (c) Spiperone binding shown in the three different lipid systems at 0.5 mg/mL D2L, and (d) spiperone binding shown in the three different lipid systems at 2.2 mg/mL D2L. A control sample which contained only soluble D2L protein and no lipid is shown in blue, to show the receptor is functional in this assay. Assay was taken at t = 0. correctly, at least in the cytoplasmic region which can bind the ligand and trigger downstream signaling. 2.4. Small Angle X-ray Scattering (SAXS). 2.4.1. Sample Preparation for SAXS. High-throughput robotic protocols were used to prepare samples for SAXS analysis based on the method provided in Darmanin et al.46 To produce lipid mixtures, known quantities of each lipid were weighed and mixed, and the dry lipids were dissolved in ethanol. Note that mol % for cholesterol was calculated based on the total mass of lipid. For mixtures containing cholesterol, chloroform was added as needed dropwise. After evaporation of the solvent in the
fume hood for 2 days, each lipid mixture was redissolved in ethanol at 200 mg/mL. A Mosquito robot (TTP Labtech, Melbourne, UK) was used to dispense 210 μg of lipid into each individual subwell of a SD-2 96-well plate. The plates were kept under a vacuum for a period of at least 24 h to remove solvent producing a thin film of lipid in each subwell. A total of 0.14 μL of the protein solution (concentration in the range 0.5−7.8 mg/mL) was then dispensed directly onto this film producing a lipidic mesophase with protein encapsulated in the ratio 60:40 (w/v) of lipid/aqueous solution. Before plate sealing, 30 μL of water was additionally dispensed into each reservoir to ensure 5016
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retention of hydration within the plates. All data were collected after a minimum of 1 h equilibration unless otherwise stated. 2.4.2. SAXS Data Collection. SAXS data, used to determine mesophase structure, were collected at the SAXS/WAXS beamline at the Australian Synchrotron. Data were collected at 2.5 and 5 h after setup of the plates. A 21.0 keV X-ray beam (wavelength = 1.033 Å) with dimensions 250 μm × 120 μm was used. The sample-to-detector distance was 1100 mm. A Pilatus 1 M detector was used to record images. The custom-built program, AXcess, was used to analyze SAXS data.48
3. RESULTS 3.1. Effect of LCP on Functionality of the Dopamine 2 Long Receptor. We assessed the functionality of the D2L receptor with respect to the cubic phase formed by three different LCP lipids: monoolein, phytantriol, and phytanoyl monoethanolamide. The typical bilayer thickness and water channel diameter for the QIID phase formed by these lipids at room temperature under excess water conditions (calculated using the method outlined in ref 36) are presented in Table 1.36 A more complete description of the calculation is provided in the Supporting Information. The D2L protein concentration ranged from 0.5−7.8 mg/ mL. Radiolabeled spiperone binding assays were used to assess the increase in receptor functionality within the lipidic cubic phase. SAXS was used to assess the corresponding structural changes to the lipidic cubic phase following protein incorporation. 3.1.1. D2L Functionality within the Monoolein LCP. Monoolein is the most commonly used lipid in LCP crystallization and has the largest water channel size and bilayer thickness of the three lipids used in this study (Table 1). Spiperone binding studies indicate that the amount of functional receptor contained within the MO lipidic cubic phase depended strongly on the protein concentration, Figure 2a,b. The highest amount of functional receptor was at 0.5 mg/ mL D2L receptor in MO with up to 90% of the total receptor reconstituting in a functional state (Figure 2a,b), producing a 586-fold increase with respect to the soluble D2L sample at the same concentration (Figure 2a). However, at higher protein concentrations the amount of functional receptor drops sharply; by 1.1 mg/mL D2L the amount of functional receptor has decreased by 550-fold (less than 0.1% of the receptor is functional) when compared to the 0.5 mg/mL concentration in MO. These binding results are a true effect of the receptor uptake into the lipid system and not the assay itself as the saturation binding curves, presented in Figure 3a, indicate that the D2L receptor is completely saturated by 0.5 mg/mL. At higher concentrations no further binding occurs, presumably because no more binding pockets are accessible to the ligand. MO, in the absence of protein, has nonspecific binding and therefore does not interfere with the assay results, Figure 3d. We therefore suggest that the large differences in spiperone binding, shown in Figure 2, are as a direct result of interactions between the receptor and the lipidic cubic phase environment and comment on this in detail in the Discussion section. Synchrotron SAXS was used to characterize the effect of protein uptake on the nanostructure of the lipidic cubic phase (Figures 4 and 5). Under the influence of the buffer components (in the absence of D2L receptor) MO adopted a diamond lipidic cubic phase (QIID) phase of lattice parameter 100 Å, similar to the MO−water system under these conditions. Peaks corresponding to the QIID phase, Figure 5a,
Figure 3. Dopamine 2 long (D2L) receptor saturation binding curves. Concentration of D2L was varied for three different lipid systems. (a) Monoolein, (b) phytantriol, (c) phytanoyl ethanomide, and (d) is a representative curve to show nonspecific binding occurs in the lipid systems when no protein is present in the assay for both monoolein (purple) and phytanoyl ethanomide (green). The solid lines represent nonspecific binding, and the dashed lines are the total binding.
are sharp and well-defined, indicating that the internal cubic architecture is well retained following addition of buffer components. Upon addition of D2L at 0.5 mg/mL the cubic phase swelled; an increase in lattice parameter of 4 Å from 100 Å to 104 Å was observed (Figure 4a). Simultaneously, diffraction peaks begin to become less well-defined, Figure 5b, indicating a loss of some long-range structural order within the cubic mesophase. As the concentration of D2L increases further, diffraction peaks become increasingly diffuse and smeared out, Figure 5c, and the cubic lattice continues to swell slightly (Figure 4a). By 4.4 mg/mL sharp diffraction peaks have been replaced by a low-intensity, diffuse ring in the diffraction pattern, indicating that the initial lipidic cubic mesophase has been replaced by an unstructured lipidic material, potentially micelles (Figure 5d). 3.1.2. D2L Functionality within the Phytantriol LCP. Phytantriol (PT) has a smaller bilayer thickness and water channel size (Table 1) compared to that of MO. The branched, isoprenoid-type hydrocarbon chain is associated with a significantly higher bilayer lateral pressure49 which can impact protein functionality and uptake.50 Similar to MO, the highest amount of functional receptor occurred at a protein concentration of 0.5 mg/mL D2L receptor, producing a 182fold increase with respect to the soluble D2L sample at the same concentration, relating to a 30% increase in functional D2L in the sample (Figure 2a,b). At higher receptor concentrations the amount of functional receptor again drops significantly, becoming of similar magnitude to that expected for detergent solubilized D2L. We discuss potential reasons for this effect in the Discussion. SAXS results indicated that the response of the PT LCP structure to protein incorporation was similar to that of MO, Figure 4b. A small increase in the lattice parameter of the diamond lipidic cubic phase was observed up to 2.2 mg/mL D2L (Figure 4b). At the same time diffraction peaks corresponding to the lipidic cubic phase become less welldefined and highly spotty, Supporting Information, Figure S1. By 2.2 mg/mL several samples already showed no distinct Bragg diffraction peaks, and by 4.4 mg/mL no Bragg peaks 5017
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of functional receptor within a lipid mesophase environment was significantly higher than that for the detergent solubilized protein. However, at higher protein concentrations (≥1.1 mg/ mL) the amount of functional receptor within the LCP was of similar magnitude to that of the detergent solubilized sample. The amount of functional receptor varied greatly between the different lipid systems. At 0.5 mg/mL, when the amount of functional receptor is highest, MO has the highest amount of functional receptor with a 586-fold increase relative to the solubilized sample, Figure 2b. PE and PT both show a less significant increase relative to the solubilized sample. This may reflect difference in bilayer thickness and lateral bilayer pressure for these systems and is discussed further in the Discussion. At 2.2 mg/mL there was no significant difference in the amount of functional receptor between the three different lipid systems and the solubilized sample, Figure 2c. We believe this effect reflects fundamental changes to the LCP structure following protein incorporation and discuss this further in the Discussion. 3.2. Effect of Protein Incubation Time within the LCP on Receptor Functionality. The amount of functional D2L receptor encapsulated within the LCP formed by MO, PT, and PE was monitored via spiperone binding measurements at time intervals of 0, 1, 2.5, and 4 h, Figure 6. Experiments were carried out at 0.5 mg/mL D2L receptor, where the amount of functional D2L in the lipidic phase is at its highest, and at 2.2 mg/mL, where the amount of functional D2L in the lipidic phase is similar to the solubilized D2L sample. At 0.5 mg/mL optimal functionality of D2L receptor was achieved between 1 and 4 h for all lipids, Figure 6a. The time to achieve optimum functionality varied between the three lipids: PE (1 h incubation); PT (2.5 h incubation); MO (4 h incubation). The need to incubate the protein within the lipidic mesophase for several hours has previously been suggested by Wallace et al.51 Interestingly, at 2.2 mg/mL, when the receptor functionality is low, no significant increase in functionality with time is observed, Figure 6b. Note that the soluble D2L sample itself (with no lipid present) was only viable for 1 h after deposition onto the plate surface. SAXS data were also observed to vary slightly with time. Specifically, data obtained 2.5 h after setup were slightly less reproducible (Supporting Information, Figure S2) than those obtained 5 h after setup (Figure 4), indicating that the structure of the LCP takes several hours to equilibrate following protein incorporation.
Figure 4. The change in cubic lattice parameter for two lipid systems with increasing dopamine 2 long (D2L) receptor concentration. The concentration of D2L was varied for two different lipid systems (a) monoolein and (b) phytantriol. Small angle X-ray scattering data were obtained 5 h after setup of the plates.
were observed for any of the samples indicating a complete loss of the cubic architecture. 3.1.3. Phytanoyl Monoethanomide. Phytanoyl monoethanomide (PE) contains the same branched C16 chain as PT, but with a monoethanolamide headgroup. The water channel size and bilayer thickness for the PE LCP are similar to that of PT (Figure 1 and Table 1). The amount of functional receptor varied with receptor concentration in a manner similar to that in MO and PT (Figure 2a,b). The highest amount of functional receptor was observed at 0.5 mg/mL; a 63-fold increase in receptor functionality (10% increase in total receptor functionality) was observed with respect to the soluble D2L sample at the same concentration. As for the other lipids the percentage of functional receptor decreased with increasing protein concentration. Because of low amounts of available lipid, no SAXS data were collected for D2L incorporated within the cubic phase of PE. 3.1.4. Overall Effect of Lipid on Amount of Functional Receptor. A similar trend with increasing protein concentration was observed for all three LCP systems studied. The amount of functional receptor was highest at 0.5 mg/mL D2L, the lowest receptor concentration studied, and decreased significantly at higher protein concentrations. At 0.5 mg/mL D2L the amount
4. DISCUSSION The effect of the lipidic cubic phase on crystal growth during in meso crystallization remains poorly understood. However, recent studies have shown that the lipid mesophase does not act as an inert matrix. Encapsulation of the protein has been shown to destroy the underlying cubic architecture in many cases,52 potentially rendering the lipid matrix unsuitable for crystal growth. The effect tends to be concentration dependent; the cubic phase is able to encapsulate low concentrations of protein, but the structure is lost at higher protein concentrations.36,37,53 It is also strongly dependent on a geometric mismatch between the hydrophilic and transmembrane domains of the protein, and the aqueous channel size and bilayer thickness of the cubic phase, respectively. Previous research has suggested that the structure of the lipidic cubic phase can impact the conformation50 and thermal stability54 of the encapsulated protein. Herein we show that encapsulation of protein within the lipid bilayer is associated with a significant increase in the amount of functional receptor for all three lipids, 5018
DOI: 10.1021/acs.cgd.6b00576 Cryst. Growth Des. 2016, 16, 5014−5022
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Figure 5. 2-D SAXS images of monoolein with different concentrations of dopamine 2 long (D2L) receptor. (a) Buffer control sample, (b) 0.5 mg/ mL D2L, (c) 2.2 mg/mL D2L, and (d) 4.4 mg/mL D2L. Note that the diffuse peak to wide angle is scattering from the UV transparent polymer which makes up the SD-2 96-well plate.
We note that the cubic phase architecture is destroyed at relatively low protein concentrations (>0.5 mg/mL). This has been previously observed for other GPCRs including the butyrate receptors.39 While previous research on the D2L receptor found retention of the cubic phase up to much higher concentrations, we have previously shown that the effect of the D2L receptor is highly batch dependent and may reflect intrinsic heterogeneities in different batches, including with bound membrane lipids. The destruction of the cubic phase nanostructure at relatively low protein concentrations is particularly common for proteins with large unstructured hydrophilic (extra- or intracellular) domains, as for the D2L receptor studied herein.36,37,39,58 These intracellular domains are typically highly flexible, unstructured loop regions which may prevent crystallization for such proteins at higher protein concentrations. We have observed a similar effect in a recent study comparing the effects of bacteriorhodopsin, with the D2L receptor which contains a large hydrophilic loop. While the D2L receptor destroyed the underlying cubic matrix at low protein concentration, the mainly hydrophobic protein, bacteriorhodopsin, had limited effect on the cubic phase structure up to much higher protein concentrations36,37 This is consistent with the observation that the majority of GPCRs which have been crystallized to date in meso have had their large third intracellular loop region removed and/or modified with an addition of a modified T4-lysozyme protein to aid crystallization. T4-Lysozyme is a highly ordered protein that aids the crystallization process by inducing the crystal contact for crystal growth. Successful crystal growth is therefore more
compared to detergent solubilized protein. The initial reconstitution of the protein within the LCP is important for successful crystal growth; retention of the LCP bilayer structure after protein addition is of particular importance for protein functionality. In addition we note that typical crystallization conditions (low MW PEG, salt concentration
