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Protein Expression and Purification
Cell Lines: DH10Bac, Spodoptera frugiperda (Sf9), Expi293FTM Cells (Thermo-Fisher, Homo sapiens)
Vector: pFB-LIC-Bse (Sf9 Expression), pHTBV1.1-LIC (Expi293 Expression)
Tags and Additions: C-terminal TEV cleavable 10His-FLAG Tag.
TMEM16K Construct Sequence:
MKVTLSALDTSESSFTPLVVIELAQDVKEETKEWLKNRIIAKKKDGGAQLLFRPLLNKYEQETLENQNLYLVGASKIRMLLGAEAVGLVKECNDNTMRAFTYRTRQNFKGFDDNNDDFLTMAECQFIIKHELENLRAKDEKMIPGYPQAKLYPGKSLLRRLLTSGIVIQVFPLHDSEALKKLEDTWYTRFALKYQPIDSIRGYFGETIALYFGFLEYFTFALIPMAVIGLPYYLFVWEDYDKYVIFASFNLIWSTVILELWKRGCANMTYRWGTLLMKRKFEEPRPGFHGVLGINSITGKEEPLYPSYKRQLRIYLVSLPFVCLCLYFSLYVMMIYFDMEVWALGLHENSGSEWTSVLLYVPSIIYAIVIEIMNRLYRYAAEFLTSWENHRLESAYQNHLILKVLVFNFLNCFASLFYIAFVLKDMKLLRQSLATLLITSQILNQIMESFLPYWLQRKHGVRVKRKVQALKADIDATLYEQVILEKEMGTYLGTFDDYLELFLQFGYVSLFSCVYPLAAAFAVLNNFTEVNSDALKMCRVFKRPFSEPSANIGVWQLAFETMSVISVVTNCALIGMSPQVNAVFPESKADLILIVVAVEHALLALKFILAFAIPDKPRHIQMKLARLEFESLEALKQQQMKLVTENLKEEPMESGKEKATAENLYFQSHHHHHHHHHHDYKDDDDK
(underlined sequence contains vector encoded TEV protease cleavage site, His and FLAG tag)
TMEM16K Construct Design and Cloning
The Homo sapiens TMEM16K gene, which encodes the TMEM16K/anoctamin-10 protein, was provided by the DNASU Plasmid collection. Coding DNA for the full length human TMEM16K sequence (NM_018075), Met1 to Thr660 (Uniprot ID: Q9NW15), was cloned into the baculovirus transfer vector pFB-CT10HF-LIC (available from The Addgene Nonprofit Plasmid Repository) for expression in Spodoptera frugiperda (Sf9) cells (Thermo-Fisher Scientific, Cat. No. 11496015). The vector adds a C-terminal TEV-cleavable His10-FLAG tag for purification. For mammalian expression, the same construct was also cloned into the pHTBV1.1-LIC baculovirus transfer vector (The BacMam vector backbone (pHTBV1.1), which was kindly provided by Professor Frederick Boyce, Massachusetts General Hospital, Cambridge, MA and adapted for ligation independent cloning in house) for expression in Expi239F cells (Thermo-Fisher Scientific, Cat. No. A14527). This vector also adds a TEV cleavable His10-FLAG tag to the C-terminus of the protein.
TMEM16K Expression
For both Sf9 and Expi293F Expression, baculoviral DNA, produced by transformation of DH10Bac with either the TMEM16K-pFB-CT10HF-LIC or TMEM16K-pHTBV1.1-LIC transfer vectors, were used to transfect Sf9 cells to produce baculovirus particles for transduction. Virus was amplified by transducing mid-log Sf9 cells (2x106 cells ml-1) grown in Sf900IITM media with 2% fetal bovine serum. Cells were incubated on an orbital shaker for 65 hours at 27°C in 1 L shaker flasks. Baculovirus were harvested by centrifugation at 900 x g for 20 mins and the virus containing supernatant was used to infect 1 L of mid-log phase (2x106 cells ml-1) cultures of Sf9 cells in Sf-900™ II Serum Free Medium (Gibco/Thermo-Fisher) in a 3 L flask, which were then grown for 72 hours at 27 °C on an orbital shaker. Cells were harvested by centrifugation at 900 x g for 15 mins, washed with Phosphate Buffered Saline (PBS), and pelleted again prior to flash freezing in liquid N2, then stored at -80 °C until needed. For mammalian (Expi293F) expression, baculovirus were prepared in Sf9 cells as described for insect cell expression. 1L of Expi293F cell cultures (2 x 106 cells ml-1) in Freestyle 293TM Expression Medium (Thermo-Fisher) were infected with high-titre P3 baculovirus (3% v/v) in the presence of 5mM sodium butyrate in a 2L roller bottle (Biofil). Cells were grown in a humidity controlled orbital shaker for 48 hours at 37 °C with 8% CO2 before being harvested by the same process as for insect cells.
TMEM16K Cell Lysis and Solubilisation
Buffer A: 20 mM HEPES pH 7.5, 200 mM NaCl, 5 % glycerol v/v, 2 mM tris(2-carboxyethyl)phosphine (TCEP)
Sf9 or Expi293F cell pellets containing heterologously overexpressed protein destined for crystallographic analysis were resuspended in 30 ml / L equiv. original cell culture Buffer A and lysed by two passes through a Emulsiflex C5 homogeniser (Avestin). A 10:1 mixture of n-Undecyl-β-D-Maltopyranoside (UDM) / cholesteryl hemisuccinate (CHS) was added to the lysate, giving a final concentration of 1 % (w/v) for UDM and 0.1% for CHS, and incubated at 4 °C for 1 hour on a roller. Insoluble material was removed by centrifugation at 32,000 x g for 1 hour at 4 °C.
Immobilised Metal Affinity Chromatography and Gel Filtration Purification
Column 1: 50% slurry TalonTM Co2+-Resin (1ml / L Original Cell Culture) in gravity column
Column 2: Superose 6 10/300 Size Exclusion Chromatography Column
The supernatant was supplemented with imidazole, pH 7.5 to a final concentration of 5 mM, and then a 50% slurry (v/v) pre-equilibrated TalonTM resin (1 mL of slurry per L original culture volume) was added. The suspension was then incubated at 4 °C on a roller for 1 hr. Talon resin was collected by centrifugation at 900 x g for 15 min and transferred to a gravity column where the remaining liquid was allowed to flow through. Subsequently, all buffer solutions were supplemented with 0.045%:0.0045% (w/v) UDM/CHS. The resin was washed with 25 column volumes of Buffer A with 20 mM imidazole, pH 7.5. TMEM16K was eluted with Buffer A + 250 mM imidazole. Peak elutions were then exchanged into Buffer A using Sephadex PD-10 desalting columns (GE Life Sciences). The C-terminal 10xHistidine-FLAG tag was removed by overnight incubation with 5:1 (w/w) TMEM16K: Tobacco Edge Virus (TEV) protease. TEV protease and contaminants were removed by adding 1.5 mL pre-equilibrated 50% Talon resin and batch binding at 4 °C for 1 hour on a rotator. The resin was removed by passing over a gravity column, with the resulting flow-through containing cleaved TMEM16K. Cleaved TMEM16K was concentrated to <1mL using a Vivaspin 20 centrifugal concentrator (GE Life Sciences) and further purified by size exclusion chromatography on a Superose 6 size exclusion column in Buffer A.
Structure Determination
Crystallisation
Purified TMEM16K was concentrated to 10-30 mg/ml using a Vivaspin 20 centrifugal concentrator with a 100 kDa molecular weight cut-off. Protein concentration was determined from the A280 using a Nanodrop spectrophotometer. TMEM16K was crystallised using both sitting drop vapour diffusion (VD) crystallisation and in meso in the lipidic cubic phase (LCP). VD crystals were grown in 0.1 M HEPES pH 7.0, 0.1 M calcium acetate, 22 % (v/v) PEG400, 0.05 mM C12E9 at a protein concentration of 10 mg/ml. For LCP crystallisation, 30 mg/ml TMEM16K was combined with 1-(7Z-hexadecenoyl)-rac-glycerol (monoacyl-glycerol 7.9, Avanti Lipids) in a 1:1.5 ratio to form a lipid cubic phase. A 50nl bolus of LCP-reconstituted TMEM16K was dispensed onto a glass LCP plate (Marienfeld, Germany) and overlaid with 800 nl of crystallisation solution. TMEM16K crystals grew in a LCP in a mother liquor containing 0.1 M MES pH 6.0, 0.1 M NaCl, 0.1 M CaCl2, 30 % (v/v) PEG300. For both LCP and VD crystallisation, initial crystals appeared after 1 week and grew to full size within 3-4 weeks.
X-ray data collection and structure determination
All X-ray diffraction data were collected on the I24 microfocus beamline at the Diamond Light Source (Didcot, UK) from single crystals using a fine phi slicing strategy. Intensities were processed and integrated using XDS (14) and scaled using AIMLESS (15). The initial dataset collected on the LCP derived crystals was phased by molecular replacement using PHASER (16) with the nhTMEM16 structure (PDB: 4WIS) as an initial search model. Phase improvement was performed using phenix.mr.rosetta (17). The final model was built using COOT (18) and refined using BUSTER (19) using all data to 3.2 Å with appropriate NCS restraints. The final LCP model was subsequently used as a starting model for molecular replacement to solve the structure of detergent-solubilised TMEM16K using an anisotropic dataset collected from a crystal grown using sitting-drop vapour diffusion methods. The anisotropic nature of the VD dataset prevented it being solved using nhTMEM16 (the only high-resolution structure at the time) as a molecular replacement search model. Processing of the VD dataset was similar to that for the LCP crystals, with additional anisotropy correction performed using STARANISO (20). The model geometry of the VD dataset was improved by using LSSR target restraints to the LCP structure during BUSTER refinement in addition to TLS and NCS restraints.
The final LCP model encompasses residues Ser14 to Gln639. Several loops were poorly order and not modelled; residues 57-67 and part of the α7-α8 loop (residues 472-474) were disordered in chain A. The loops connecting either α5 and α6 or TM3-TM4 were poorly defined in both chains of the dimer and have also not been modelled. The final model also includes three Ca2+ ions per monomer along with an additional Ca2+ at the N-terminal end of TM10 on the dimer axis. The presence of Ca2+ ions at these sites was indicated by peaks in both anomalous difference and PHASER log-likelihood gradient (LLG) maps calculated using a 3.4 Å dataset with high multiplicity collected at a wavelength of 1.65 Å. Two Ca2+ ions in each dimer lie at the canonical two Ca2+ ion binding site and a third lies at the junction of TM10 and α10. All of these ions have bonds that are less than 2.5 Å to sidechains, suggesting the ions are not hydrated. The fourth Ca2+ ion identified in the anomalous difference maps lies on the dimer 2-fold axis, binding to the backbone of the ER loop between TM9 and 10. The greater than 4 Å interaction distances suggest that this ion is hydrated and is likely to be the result of the high (100 mM) [Ca2+] used in the crystallisation conditions. This fourth ion is only present in the LCP dataset, not in the vapour diffusion structure, so the presence of this ion is not necessary for the open conformation to be formed. In addition, elongated density within the dimer interface were interpreted as MAG7.9 lipids.
Cryo-EM grid preparation
3 μl aliquots of TMEM16K protein purified in UDM/CHS, at a concentration of 5 mg/ml was either applied to grid directly (430nM Ca2+ samples) or supplemented with 2 mM CaCl2 or 10 mM EGTA, then were applied to glow-discharged holey carbon grids (Quantifoil R 1.2/1.3 Cu 300 mesh). Grids were blotted at 80-100% humidity for 3-5 s at 5 °C and plunge-frozen in liquid ethane using a Vitrobot Mark IV, (FEI). Ca2+-free TMEM16K grids were prepared in an identical manner using protein solution supplemented with 10 mM EGTA.
Cryo-EM structure determination methods
All initial processing was carried out in RELION 2.0 (21) & 3.0 (22). Frames in each movie stack were aligned and dose-weighted with MotionCor2 (23). CTF parameters were estimated using CTFFIND 4.0 (24). Dose weighted stacks were subjected to semi-automatic particle picking using Gautomatch. Particles were picked from a subset of micrographs and 2D classified to produce class averages. The resultant representative class averages were then used as templates in Gautomatch for autopicking the full image sets. Particles were subjected to multiple (5-6) rounds of reference-free 2D classification. An initial model was generated ab initio in RELION and used as a reference of 3D classification with no symmetry imposed. Particles belonging to the best / highest resolution class(es) were pooled and taken forward into a second round of 3D classification with C2 symmetry. Finally, particles from the highest resolution class were used for auto-refinement. The 2 mM and 500 nM datasets were further processed in RELION 3 (22) to take advantage of new particle polishing and CTF refinement routines. In both cases, two rounds of individual particle CTF refinement interspersed with a single step of Bayesian polishing and 3D auto-refinement produced the best maps. Further static 3D classification where no image alignment was performed with multiple (10) classes yielded a slightly better resolved reconstruction for the 2 mM CaCl2 dataset. Local resolution estimation for each final reconstruction was performed with RELION (25). Post-processing was carried out in RELION using a mask extended by 12 pixels with an additional 12-pixel soft edge that excluded the detergent micelle surrounding the protein. Conformational homogeneity appears to be well maintained within each dataset and at no stage during 3D classification could we detect a subset of particles displaying a more open groove conformation. For the most part, TM3 and TM4 remain well resolved within and between different 3D classes indicating a lack of structural heterogeneity for the region that is responsible for defining the extent of the scramblase groove.
Model building was carried out manually using the 3.4 Å TMEM16K LCP crystal structure (PDB: 5OC9) as a template. Briefly, chain A of the crystal structure was roughly fitted into the 3.5 Å resolution 2 mM Ca2+ post-processed cryo-EM map in UCSF Chimera (26). Subsequent model building was carried out in COOT (18). The cytoplasmic domain was rotated into density and then appropriate sub-regions were fit to the density as rigid bodies. TM helices were fitted as rigid units or segmented where appropriate. The C-terminal α10 helix was rotated manually into position and side chains were fitted using preferred rotamers. The remodelled chain A was superposed onto chain B and globally fitted to the cryo-EM density to create the symmetric dimer. Refinement was carried out at 3.5 Å resolution against the post-processed RELION map (low pass filtered to 3.47 Å and sharpened with a B-factor of -112 Å2) using phenix.real_space_refine (PHENIX v1.14) using NCS constraints, rotamerrestraints along with secondary structure restraints. Missing loop regions, not modelled in the crystal structure, were added and the calcium coordination was initially defined using phenix.metal-coordination. Subsequently the calcium coordination at both sites was maintained using distance restraints derived from the LCP X-ray structure. The final model encompasses all residues from Ser13-Lys641 and three Ca2+ ions per monomer. Some of the lipid / detergent like density around the TM domain at the dimer interface was modelled by six UDM molecules per monomer and a single phosphatidylcholine lipid. The precise identity of the lipid bound at the dimer interface between TM3, 5 and TM10 (of the adjacent monomer) is unknown.
The structure of the 430 nM Ca2+ complex is very similar to the 2 mM Ca2+ structure and only required minor adjustments to the poorly-resolved loop regions between α5-α6 and α7- α8. The density for the detergent and lipid molecules was present in the map but less convincing at this resolution and so these heterogroups were removed. The resultant model was refined against the 4.2Å RELION post-processed map (sharpened with a B-factor of -176Å2) using phenix.real_space_refine. Model geometry was maintained using NCS constraints in combination with reference model restraints to the 2 mM Ca2+ structure.
The 2 mM Ca2+ structure also served as a template for the low resolution Ca2+-free structure. The 2 mM Ca2+ model was initially docked into the map using Chimera and then refined using phenix.real_space_refine with global minimisation and morphing using default restraints/constraints and additional secondary structure restraints. A cryo-EM map, low-pass filtered to 5.1 Å resolution and sharpened with a B-factor of -150 Å2, was used for all refinement and model building. The TM1-loop-TM2 region (residues 222-254) and the N-terminal end of TM10 required additional manual rebuilding/fitting in COOT. The cytoplasmic α7- α8 region was poorly resolved and truncated between residues 463-474. At this resolution, there was no obvious density for the amino acid sidechains and the vast majority were truncated to their C-beta atoms. Only the sidechains of prolines and a few large hydrophobic residues were retained. In addition, the Ca2+ ions were removed from the model. The resultant model was further refined with phenix.real_space_refine using NCS constraints, secondary structure restraints and reference model restraints to the 3.5 Å 2 mM Ca2+ structure. All structures / refinement protocols were validated by randomising the final models by applying coordinate shifts of up to 0.3 Å using the noise function in PDBSET (CCP4). The resultant shifted models were then refined against the corresponding post-processed half1 maps. Model-to-map Fourier shell correlations (FSCs) were then calculated with phenix.mtriage using either the final refined model against the full map (FSCsum) or the randomised half map1 refined model against either the half1 map (FSCwork) or the half2 map (FSCfree) not used in the validation refinement.
Functional Assays
Liposome reconstitution and lipid scrambling assay
Liposomes were prepared, as described previously (27), from a 7:3 mixture of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) 1-palmitoyl-2-oleoyl-glycero-3-phosphoglycerol (POPG). Lipids in chloroform (Avanti), including 0.4% w/w tail labelled NBD-PE, were dried under N2, washed with pentane and resuspended at 20 mg ml-1 in buffer B (150 mM KCl, 50 mM HEPES pH 7.4) with 35 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). TMEM16K was added at 5 µg protein/mg lipids and detergent was removed using five changes of 150 mg mL-1 Bio-Beads SM-2 (Bio-Rad) with rotation at 4°C. Calcium or EGTA were introduced using sonicate, freeze, and thaw cycles. Liposomes were extruded through a 400 nm membrane and 20 µL were added to a final volume of 2 mL of buffer B supplemented with 0.5 or 2 mM CaCl2 or 2 mM EGTA. The fluorescence intensity of the NBD (Excitation: 470 nm, Emission-530 nm) was monitored over time with mixing in a PTI spectrophotometer and after 100 seconds sodium dithionite was added at a final concentration of 40 mM. Data was collected using the FelixGX 4.1.0 software at a sampling rate of 3 Hz.
Bulk Flux Assay
Cl− flux assay was conducted as described previously (28). Liposomes were prepared as per the lipid scramblase assay and equilibrated in external buffer with low KCl (1 mM KCl, 300 mM Na-glutamate, 50 mM HEPES, pH 7.4) by spinning through a Sephadex G50 column (Sigma-Aldrich) pre-equilibrated in external buffer. To complete the experiment, 0.2 mL of the flow through from the G50 column was added to1.8 ml of external solution and the total Cl− content of the liposomes was measured using an Ag:AgCl electrode after disruption of the vesicle by addition of 40 μL of 1.5 M n-octyl-β-D- glucopyranoside (Anatrace). The fraction of liposomes containing at least one active TMEM16K ion channel, A, was quantified as follows:
where ΔCl is the change in [Cl−] recorded upon detergent addition in protein-containing vesicles and ΔClPF is the Cl− content of protein-free liposomes prepared in the same lipid composition on the same day.
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