Puneet Juneja, Wolfram Welte
Department of Biology, University of Konstanz, Konstanz, Germany
Manuscript in Preparation
2.1 Abstract
Detergents and lipids are critical for oligomerization, function and crystallization of membrane proteins. The Torpedo nicotinic acetylcholine receptor (nAChR) has been widely studied in the last decades but its inability to crystallize it has moved focus towards more stable protein preparations, new detergents and lipid based crystallization methods. Lipid analoge detergents and lipids have shown to preserve structure and function of the nAChR. In the present work we introduce a new efficient method for affinity purification of Torpedo nAChR as a complex with alpha-bungarotoxin. The oligomeric stability of affinity purified nAChR- alpha-bungarotoxin complex was analyzed on size exclusion chromatography (SEC) in the presence of different detergents. We introduce lipid analoge detergents, dodecanoyl-propanediol-3-phoshorylcholine (ET12H), 1-dodeoxy-propanediol-3-phophorylcholine (ES12H) and non-lipid analog trans-4 (trans-4’-alkylcyclohexyl) cyclohexyl-a-D-maltoside (PCC-α-M), for purification of the Torpedo nAChR.
Here we demonstrate that Torpedo californica nAChR in complex with alpha-bungarotoxin in different detergents preserve its native dimeric structure.
Key words: detergents, nAChR, lipids, oligomerization, dimer, alpha-bungarotoxin
2.2 Introduction
Nicotinic acetylcholine receptors (nAChRs) are members of the Cysteine loop receptor family (CLR). They are crucial for cholinergic pathways in the neuronal system and are fundamental for synaptic transmissions and regulation. In mammals they occur in neuronal and neuromuscular junctions. Dysfunction of nAChRs has been implicated in many neurodegenerative diseases (72, 73). nAChR from the Torpedo electric ray is a homologue of the muscle type nAChR. It has heteropentameric architecture (α2βγδ) with five subunits forming a cation selective channel. Each subunit consists of an extracellular domain, four transmembrane helices TM1, TM2, TM3, TM4, and a large cytoplasmic loop between TM3-TM4. Heteropentameric nAChRs are difficult to express recombinantly and native sources like Torpedo electric rays such as Torpedo californica and Torpedo marmorata electric organ have been a prominent source of nAChRs for biophysical and structural studies. An electron microscopic structure of nAChR at 4 Å is available from Torpedo marmorata membranes (74–78). A significant progress has been made in the last decade in determining crystal structure of CLRs. A high resolution structure of AChBP (acetyl choline binding protein) from snail Lymnaea stagnalis (79), a homologue of nAChR provided first insights into extracellular domain architecture. Crystal structures of bacterial homologues GLIC (80) and ELIC (81) showed the organization of transmembrane helices. The first crystal structure of an eukaryotic CLR was of the glutamate gated ion channel (GluCl) from C. elegans with a truncated TM3-TM4 loop (45), both bacterial and eukaryotic CLR crystal structures show that lipids are critical for crystallization. The bacterial homologue GLIC was crystallized in open conformation with 6 DDM molecules and 12 lipid molecules (named LIP601/2/3) (34), the GLuCl could be crystallized only in presence of lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (45). These observations show the importance of detergents and lipids in the crystallization of full-length Cysteine loop receptor.
Inability to crystallize Torpedo nAChR could be attributed to a number of reasons; Extensive glycosylation, impurities, lack of suitable detergents and conformational inhomogeneity. The nAChR are heteropentamers with pseudo five fold symmetry and are found as dimers of pentamers in cell membranes. It has been shown that nAChR dimerize by forming cystines from adjacent δ subunit (82, 83). Recent work by (84, 85) has shown also that lipids plays an important role in the oligomeric state of the nAChR (monomer, dimer and aggregates) and differential loss of lipids during ligand (bromoacetylchloline) affinity purification in different detergents results in different ratios of oligomers when checked with SEC. Other studies on Torpedo nAChR showed a minimum
requirement of phospholipids for channel activity of at least 20 mole of lipids per mole protein and 45 moles for full channel activity (86, 87). Most studies indicate the loss of channel activity upon lipid depletion (87).
As compared to the agonist bromoacetylchloline used by others (84, 87), we used the antagonist alpha-bungarotoxin to construct an affinity column to evaluate the stability of purified Torpedo nAChR. We describe here alpha-bungarotoxin affinity purification and the stability of the nAChR-BgT complex in different detergent environment using size exclusion chromatography (SEC) in lipid based detergents.
2.3 Experimental procedures Membrane preparation
Torpedo californica electric organs were purchased from Aquatic research consultants, USA. 100-gram electric organ stored at -80 °C was minced/grounded into small pieces 5-7 mm with mortar pestle in presence of liquid nitrogen. The tissue was thawed in 200 ml of homogenization buffer- 50 mM Tris, 3 mM EDTA, 2 mM EGTA pH 7.5, 10 mM iodocaetamide, 0.01% NaN3, 1mM PMSF and Ultra protease inhibitor (Roche) and further homogenized using an Omni Mixer homogenizer at 7000 rpm with 30 second pulse, 4 times with 2 minutes intervals for cooling on ice.
All steps were done on ice. The homogenate was centrifuged at 1500 rpm for 15 minutes and supernatant was collected and centrifuged at 40,000 rpm in Type 45 Ti rotor (Beckmann) for 1 hour. The pellet (nAChR membranes) was collected and suspended in 50 mM Tris, 150 mM NaCl, pH 8.2 and flash frozen and stored at -80 °C until further use.
Biotinylation of alpha-bungarotoxin
Alpha-bungarotoxin from Bungarus multicinctus (Invitrogen) was biotinylated with EZ-Link Sulpho-NHS-LC-Biotin (Thermo scientific) according to manufacturers protocol. Biotinylation was checked by Western blot with Streptactin conjugated to alkaline phosphatase.
Alpha-bungarotoxin Affinity purification
nAChR membranes (protein concentration of 4-5 mg/ml) were incubated with 200 nM of biotinylated alpha-bungarotoxin at 4 °C for 1 hour. The membranes were again centrifuged at 40,000 rpm in a Type 45 Ti rotor (Beckmann) for 1 hour to remove unbound/excess of biotinylated alpha-bungarotoxin. nAChR membranes with bound alpha-bungarotoxin were resuspended in 100
mM Na2HPO4, 150 mM NaCl, pH 7.2 at 4-5 mg/ml protein concentration and solubilized with respective detergents at concentration as in Table 1. Detergents Cymal-6 and Fos-choline-12 were from Anatrace, LFC-16 (1-palmitoyl-2-hydroxy-sn-glycerol-3-phosphocholine) from Avanti polar Lipids, PCC-α-M from Glycon, detergents ET12H and ES12H were from R. Berchtold, Biochemisches Labor, Bern (88). Detergent solubilized membranes were centrifuged at 40,000 rpm in Type 45 Ti rotor (Beckmann) for 1 hour. Supernatant was passed over 2 ml of monomeric Avidin column (Pierce), washed with 30 ml of washing buffer, 100 mM Na2HPO4, 150 mM NaCl, pH 7.2 containing detergent at twice the CMC, Table1. Protein was eluted with washing buffer containing 5 mM biotin. All steps were done on ice or in the cold room.
Size exclusion chromatography
Affinity purified nAChR was concentrated with 100 kDa cut off concentrator to 2-4 mg/ml and was subjected to gel filtration on Superose 6, 10/300GL column (24ml) in 20 mM Tris, 100 mM NaCl, pH 7.5 containing the respective detergent at twice the CMC, Table 1.
2.4 Results
Alpha-bungarotoxin Affinity purification
Alpha-bungarotoxin has a very high affinity of 1 nM for Torpedo nAChR, which was exploited effectively for affinity purification. Alpha-bungarotoxin was biotinylated on surface amine residues and incubated with nAChR membranes and bound with high affinity to the nAChR. The biotinylated nAChR-BgT complex was solubilized and passed over monomeric avidin column, which retained the nAChR-BgT complex via biotin-avidin interactions, which was subsequently eluted with free biotin. In all detergents tested (Table 1) we obtained purified protein, and four clear bands of Torpedo nAChR chains are clearly visible on SDS gel (Figure 3).
Size exclusion chromatography
The affinity-purified nAChR-Bgt complex was gelfiltrated by a Superose 6 column with a bed volume of 24 ml equilibrated with the respective detergent at a concentration twice the CMC.
A first peak at elution volume of the dimer was observed, followed by a second partly overlapping peak or shoulder representing the monomer was observed in all detergents (Figure 2). Two distinct peaks were never observed. In the detergent PCC-α-M 89% of nAChR elutes as dimer and 11%
nAChR elutes as monomer (Figure 3). In phospholipid-based detergent Fos-choline-12 (Fos-12),
58% of nAChR in dimers and 42% nAChR monomers, in ES12H 75% nAChR dimers, 25%
nAChR monomers and in the ET12H 85% nAChR dimers and nAChR 15% monomers were observed. In LFC-16 the monomeric peak/shoulder was not distinguishable, most of the protein eluted as a dimer (Figure 2). In the non-lipid analogue detergent Cymal-6, 79 % of torpedo nAChR eluted as dimer and 21% as monomer (Figure 3). In all detergents the dimeric conformation of nAChRs was prevalent.
2.5 Discussion
Torpedo nAChRs are expressed as dimers in the cell membrane with intersubunit disulphide linkages (82). As shown by others (82, 83) when Torpedo nAChR purified by sucrose gradient or affinity purification the complex elutes as a mixture of dimers and monomers. Initially this had been attributed to the reduction of the intersubunit disulphide bond. To overcome dimer dissociation, membranes were prepared in presence of an excess of iodoacetamide to prevent any reduction with free sulphide during lysis and to rule out any role of cysteines, but still monomers were found. In Asmar-Rovira et.al, it is argued that the dimer to monomer ratio is lipid dependent and differential loss of lipids in different detergents during bromoacetylchloline affinity purification (89) results in different dimer to monomer ratios. In (84) sucrose gradient purified nAChR membranes were solubilized and purified on bromoacetylchloline bromide affinity chromatography. Purified nAChR was dialysed and binding to fluorescent alpha-bungarotoxin and (nAChR-Bgt) was used to evaluate dimer and monomer ratio using analytic SEC in presence of DDM (different detergents were used only during solublization and affinity purification).
It is evident that lipid depletion can take place during all steps of purification i.e. sucrose gradient, solubilization, affinity purification and gel filtration. Therefore we aimed for a method to purify nAChR with a minimum loss of lipids. Alpha-bungarotoxin has a very high affinity of 1 nM for Torpedo nAChR with two binding sites per nAChR molecule. In membranes nAChR exists as dimer of pentamers and the nAChR-BgT complex is itself very stable. We exploited this for an affinity purification method based on biotinylated alpha-bungarotoxin. nAChRs were first trapped as a complex with biotinylated alpha-bungarotoxin in the membranes and later solubilized, affinity purified over monomeric avidin and followed by SEC. Alpha-bungarotoxin affinity gave a highly pure (95%) protein in all the detergents (Figure 1). nAChR retains its native dimeric conformation in all detergents tested, on average 85% of nAChRs were dimeric.
In detergent PCC-α-M (90) the alkyl chain (as in maltosides based detergent) is replaced by an
alkyl bicyclohexyl group resulting in a smaller radius of curvature and larger micelles. The lower conformational flexibility of the bicyclohexyl group makes it a milder detergent. Lipid analogue detergents ET12H and ES12H with the phosphorylcholine head group are linked differently to the alkyl chain by ether and ester linkage respectively (Table 2). The phosphorylcholine head group in ES12H and ET12H is smaller than the maltoside head group, the micelles resemble LADAO micelles more than detergent with maltoside head group C12MS (88). The relative orientation of polar head group and hydrocarbon chain in ET12H and ES12H is determined by the propanediol group which have even distribution of rotational confirmation around C-C bond not by chain stacking by hydrocarbon chains as in maltoside based detergents. LFC-16 has a 16-carbon hydrocarbon chain and a similar ester bond linkage like ES12H, it also result preferentially in dimeric nAChR as per peak elution volume, the monomeric shoulder was not distinguishable. In contrast (85) had shown 80% monomers in LF-16 detergent when purified by a bromoacetylcholine affinity column. We observed maximum percentage of 42% monomers in the detergent Fos-choline-12.
Because dimers were prevalent in all tested detergents, this led us to think of a sound strategy.
First, we don’t use a sucrose gradient to purify membranes,it could prevent loss of lipids. Second, we use alpha-bungarotoxin as a ligand for trapping nAChR-BgT complex in membranes and for affinity purification. All of these factors could affect lipid loss, the nAChR dimer to monomer ratio and the detergents tested.
The crystal structure of the human beta-2 adrenergic receptor revealed bound cholesterol and a mechanism for cholesterol mediated dimerization has been suggested (91). Example from Cys loop receptors (nAChR) family are the proton sensitive chloride channel GLIC (80). It was crystallized in a closed conformation and has both lipid and detergent DDM (Dodecyl maltoside) molecules observations suggest a possible role of lipids in stabilizing one conformation.
Other methods of nAChR purification for Torpedo californica results in oligomeric heterogeneity, where as the present method provide a good alternative, as it is mild (no need of sucrose density gradient), and provide a stable homogenous native dimeric conformation of protein, in all the
detergents tested. Trapping nAChR-BgT complex in membranes before solublization may preserve some lipids, which helps in preserving oligomeric stability and could aid in the crystallization.
Tables and Figures
Table 1. Detergents used in solublization and purification of Torpedo nAChR.
Name CMC
mM(%)
Solublization Conc
Affinity and SEC (mM)
Cymal-6 0.56 mM 1% 0.05%
PCC-α-M 0.036 mM 1% 0.064
mM
ES12H 0.016% 1% 0.032%
ET12H - 1% 0.032%
Fos-choline-12
1.5 mM 1% 0.09%
LFC-16 0.004 mM 1% (20 mM) 0.01 mM
Table 2. Representation of detergents showing head groups and alkyl chain ET12H
ES12H
PCC-α-M
LFC-16
Fos-choline-12
Cymal-6
Figures
Figure 1. SDS-PAGE of purified Torpedo nAChR over size exclusion chromatography on Superose 6, 10/300GL column (24ml) in 20 mM Tris, 100 mM NaCl, pH 7.5, 0.05% Cymal-6.
Similar pattern were obtained for all other detergents.
FIGURE 2. Size exclusion chromatography elution profile of nAChR-BgT complex in different detergents on Superose 6, 10/300GL column (24ml) in 20 mM Tris, 100 mM NaCl, pH 7.5 containing the respective detergent with twice the CMC, (Table 1).
FIGURE 3. Ratio of nAChR-BgT complex eluted as dimer and monomer of nAChR in different detergents on size exclusion column, Superose 6, 10/300GL column (24ml) in 20 mM Tris, 100 mM NaCl, pH 7.5 containing the respective detergent with twice the CMC.
10 0 20 30 40 50 60 70 80 90 100
Monomer Dimer
3. Expression, Purification and Crystallization of GABAA β3 receptor from