Structural insight into the assembly of TRPV channels.

Functional Analysis of Purified TRPV2 Channel We tested several TRPV channel homologs for structural analysis and found that rat TRPV2 gave the best expression in S. cerevisiae . Through extensive screening of buffers and detergents, we determined that the maltose-neopentyl glycol (MNG) class of detergents stabilizes rat TRPV2 as a functional tetramer ( Figure S1A available online). Recently it has been found that these newly synthesized low-critical micelle concentration detergents are optimal for extraction, long-term stabilization, and structure determination of several membrane proteins ( Chae et al., 2010 ). These detergents form a large micelle belt around the TM domain of pure membrane proteins visible in EM micrographs ( Jastrzebska et al., 2013 ; Westfield et al., 2011 ). In addition, membrane proteins purified in MNG detergents display a higher molecular weight, adding approximately 60 kDa per protein monomer ( Jastrzebska et al., 2013 ). After immunoaffinity purification of TRPV2, size-exclusion chromatography (SEC) and native gel electrophoresis revealed that a homogenous sample was obtained with an approximate molecular weight of 600 kDa ( Figure S1A ). This mass accounts for the 360 kDa TRPV2 tetramer and 240 kDa of detergent mass. The cryo-EM structures of TRPV1 and TRPV4 were determined using common maltoside detergents, such as decyl-β-D-maltoside (DM) and dodecyl-β-D-maltoside (DDM) ( Moiseenkova-Bell et al., 2008 ; Shigematsu et al., 2010 ). During detergent screens, we observed that TRPV2 purified in DM or DDM exists in multiple aggregating states confirmed by native gel electrophoresis after SEC ( Figure S1B ), suggesting that TRPV2 purified in these detergents is not suitable for structural analysis. Among the thermoTRPV channels, probenecid, a uricosuric agent, has been shown to specifically activate TRPV2 ( Bang et al., 2007 ), and trivalent cations such as Gd 3+ and La 3+ are potent blockers of TRPV2-mediated currents ( Leffler et al., 2007 ). To determine the functionality of purified TRPV2, the channel was reconstituted into preformed asolectin liposomes and electrophysiological measurements were made from excised patches in the inside-out configuration. In the presence of 100 μM probenecid in the pipette, robust single-channel currents were observed, displaying very little desensitization. These currents showed a rapid block upon application of 100 μM GdCl 3 that was fully reversible during wash-off ( Figure 1A ). Currents recorded at different holding potentials showed widely differing kinetics for inward and outward currents ( Figure 1B ). While inward currents were long bursts of high-open probability and infrequent visits to the nonconducting state, the outward currents were characterized by very fast flickers ( Figure 1B ). A plot of single-channel currents as a function of membrane potential yields a conductance of 304 ± 4 pS. These results demonstrate that purified TRPV2 retains functional properties.

EXPERIMENTAL PROCEDURES TRPV2 Expression and Purification Rat

TRPV2 with a 1D4-tag was cloned into a YepM plasmid and overexpressed in BJ5457 S. cerevisiae (ATCC). TRPV2 membranes were prepared and solubilized in 0.087% w/v lauryl MNG (Anatrace), 20 mM HEPES (pH 8.0), 150 mM NaCl, 5% glycerol, 1.0 mM dithiothreitol (DTT), and 1 mM phenyl-methanesulfonylfluoride (PMSF) for 1 hr at 4°C. The insoluble fractions were pelleted by ultracentrifugation at 100,000 g , 4°C for 45 min. The soluble fraction containing TRPV2 protein was incubated overnight at 4°C with cyanogen-bromide-activated Sepharose 4B coupled with 1D4 antibody. The column was packed and washed with washing buffer consisting of 0.006% decyl MNG (Anatrace), 20 mM HEPES (pH 8.0), 150 mM NaCl, and 1 mM DTT. The TRPV2 protein was eluted with 3 mg/ml 1D4 peptide (Genescript USA) in washing buffer, concentrated, and subjected to SEC. Single-Channel Properties of Reconstituted TRPV2 Electrophysiological measurements were made by patch clamp recordings in reconstituted proteoliposomes, as described earlier ( Chakrapani et al., 2007 ; Cortes et al., 2001 ; Delcour et al., 1989 ; Velisetty and Chakrapani, 2012 ). A total of 20 mg soybean polar extract (Avanti polar lipids) in chloroform was dried in nitrogen stream and sonicated in a buffer containing 200 mM KCl and 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS) at pH 7.0. The liposomes (at 10 mg/ml concentration) were destabilized by adding 4 mM DDM and were gently agitated for 20 min. Purified TRPV2 was added to the vesicles (in 1:50 [w/w] protein to lipid ratio) and nutated for to 2 hr. Residual detergent was removed by incubation with biobeads (Bio-Rad Laboratories) overnight at 4°C. Channel-incorporated liposome suspension was then centrifuged for 2 hr at 100,000 g and the pellet was resuspended in 60 μl of KCl/MOPS buffer. A drop of the proteoliposome was placed on a glass slide and dried overnight in a desiccator at 4°C. The sample was then rehydrated with 20 μl of buffer, which yielded giant liposomes. This preparation was suitable for patch clamp recordings after ~2 hr. Currents were recorded in 150 mM KCl and 10 mM MOPS at pH 7.0. Recording pipettes were pulled from thin-walled borosilicate glass and heat polished such that they had a bath resistance of 1–2 MΩ. The pipette solution had 100 μM probenecid in a buffer containing 150 mM KCl, 2 mM CaCl 2 , and 10 mM MOPS, pH 7.0. All measurements in this study were conducted in the inside-out patch configuration with a bath solution containing 150 mM KCl, 10 mM MOPS, and pH 7.0. Experiments were performed at room temperature (20°C–22°C). Pore blockers were applied using RCS-200 fast solution exchanger (Biologic) fed by gravity. Single currents were acquired at 20 kHz and filtered at 5 kHz using Axon 200-B patch-clamp amplifier (Molecular Devices). In the presence of probenecid, most excised patches had multiple overlapping channel activities. Only those recordings with single-open channel were used for further analysis.

Cryo-EM Preparation Purified

TRPV2 was frozen onto Quantifoil R2/1 copper grids (Quantifoil Micro Tools; 400 mesh) using the Vitrobot (FEI). Data were acquired on an FEI Tecnai F20 TWIN microscope operated at 200 keV under low-dose conditions (15 electrons/ Å 2 ). Images were collected at a nominal magnification of 62,000× at various defocus values between −1.5 and −3 μm and were recorded with a Tietz TemCam-F416 complementary metal oxide semiconductor-based camera (4k × 4k). Single-Particle Reconstruction and Modeling The EMAN2 ( Tang et al., 2007 ) boxer routine was used to pick 23,051 particle images from 650 micrographs and to generate a particle stack. The stack was binned by factors of two and four, yielding pixels of 2.7 Å and 5.3 Å, respectively, for image processing. Initial estimates for microscope defocus and astigmatism parameters were calculated with CTFFIND3 ( Mindell and Grigorieff, 2003 ). The bin4 images were filtered, normalized, and band-pass filtered (120–15 Å ) prior to reference-free multivariate statistical analysis classification in IMAGIC ( van Heel et al., 1996 ). The IMAGIC C1-Startup routine was used to generate an initial 3D model without imposed symmetry. As this initial 3D model displayed approximate 4-fold symmetry, a 3D model was also generated with imposed C4 symmetry. The two IMAGIC-generated maps were used as input 3D models for angular search and refinement of the unclassified particle images with FREALIGN ( Grigorieff, 2007 ). The FREALIGN search and refinement procedure was carried out in parallel using either C1 or C4 symmetry and was first performed on the bin4 stack and subsequently on the bin2 stack. The final resolution was 19 Å at the FSC 0.5 threshold for the FREALIGN C1-refined 3D map and 15 Å at the FSC 0.5 threshold for the FREALIGN C4-refined 3D map. The FREALIGN C4 symmetrized map was used as the input 3D model for angular search and refinement with the 3D Fourier Space programs ( http://www.sickkids.ca/research/rubinstein ; Benlekbir et al., 2012 ), which incorporate the gold-standard refinement scheme ( Scheres and Chen, 2012 ). The 3D Fourier Space refinement procedure was carried out in parallel using either C1 or C4 symmetry. The final C1 3D Fourier Space refined map ( Figure S2E , related to Figure 2 ) has a resolution of 20 Å at the 0.143 threshold of the gold-standard FSC curve (25 Å at the 0.5 threshold). For processing with symmetry, C4 symmetric odd and even maps were calculated at the end of each round with FREALIGN in iflag = 0 mode. The 3D Fourier Space cross correlation (CC) values were converted into FREALIGN-style phase residuals with the following equation: phase residual = 100 – CC value. A C4 symmetric final map based on the full data set was calculated at the end of the last refinement round. The final structure is based on all 23,051 particles and has a resolution of 13.6 Å at the 0.143 threshold of the gold-standard FSC curve (19 Å at the 0.5 threshold). The final map is shown sharpened with a B-factor of −1,000 Å 2 and filtered with the C ref filter ( Rosenthal and Henderson, 2003 ). The cryo-EM structure has been deposited in the EM Data Bank (EMD-5688). The electrostatic potential surfaces for the N-terminal TRPV2 ARDs, TRPV1 ARDS, and TRPV4 ARDs were calculated using Adaptive Poisson-Boltzmann Solver ( Baker et al., 2001 ). Fitting of the TRPV1, TRPV2, TRPV4, and TRPV6 ARD crystal structures was perform using Colores in the Situs package ( Chacón and Wriggers, 2002 ). Visualizations and MlotiK1 crystal structure fitting into the TRPV2 EM map were performed using UCSF Chimera software ( Pettersen et al., 2004 ). TRPV2 Structure Hand Determination Tilt-pair cryo-electron micrographs were collected at tilt angles of 0° and 20° using an FEI Titan Krios at 120 keV and magnification of 59,000× with a 10 electron/ Å 2 dose on an FEI 4k ×4k charge-coupled device detector. A total of 186 tilt-pair particle images were selected and binned by a factor of four, yielding a pixel size of 5.4 Å for image processing. The tilt axis, defocus, and astigmatism estimations for the tilted micrographs were calculated using CTFTILT, whereas CTFFIND3 was used for the defocus and astigmatism estimations for the untilted micrographs. The orientational parameters were determined independently for both the 0° and 20° particle images using FREALIGN search and refinement procedures with imposed C4 symmetry and with a resolution range of 150–18 Å, as recommended by Henderson et al. (2011) . The parameter files for the 0° and 20° particle images served as input to Tiltdiffmulti, a program designed to determine the best tilt axis and tilt angle relating two views of a particle ( Henderson et al., 2011 ). A tilt-pair parameter plot (TPPP) was generated for TRPV2, and it reveals a cluster of points centered around a tilt axis of 270° and a tilt angle of 20° ( Figure S4 , related to Figure 2 ). A simulated tilt-pair data set was generated using a protein with a known and distinct handedness, Cand1 (PDB ID code: 1U6G; Goldenberg et al., 2004 ), as described by Williams et al. (2008) . The simulated tilt-pair data set was put through the same particle selection, search and refinement, and TPPP analysis scheme as the TRPV2 tilt-pair data set. The simulated data analysis, which was performed with a 3D search model of the correct hand, yielded a TPPP plot with a cluster centered at a tilt axis of 270° and a tilt angle of 20°, as found for TRPV2. The convergence of the clusters for the simulated and experimental data sets suggests that the hand of the TRPV2 map is correct.