Plasmids preparation
Full-length human prestin (hPres) was amplified using human universal reference cDNA (Zyagen) as the template, and cloned into a modified pEG BacMam vector with a C-terminal TEV cleavage site followed by a FLAG-EGFP tag. A thermostabilized prestin protein (hPresTS) was designed by the consensus mutagenesis approach, based on the sequences of all eukaryotic prestin orthologs (https://github.com/TaizoAyase/consensus_creator) 17. About 40% of the amino acid residues were altered in hPresTS as compared to wild-type hPres (Supplementary Fig. 2). A cDNA encoding hPresTS was synthesized with codons optimized for expression in human cell lines, and cloned into the modified pEG BacMam vector described above. For functional assays, naked mole rat (Heterocephalus glaber) prestin (HgPres) was used for better expression as compared to hPres. The amino-acid sequence of HgPres is 96% identical to that of hPres (Supplementary Fig. 2). Wild-type HgPres cDNA was synthesized based on the mRNA sequence (XM_004839711.2) by General Biosystems (Durham, NC), and cloned into the pSBtet-Pur vector 43 with a C-terminal mTurquoise2 (mTq2) tag. Standard mutagenesis methods were used to generate HgPres mutants in the pSBtet-Pur vector.
Purification of hPres and hPresTS
Baculoviruses carrying the prestin constructs were produced and amplified in Sf9 cells, using the Bac-to-Bac system (Invitrogen). HEK293S GnTI- cells (ATCC, CRL-3022) were grown and maintained in FreeStyle 293 medium (Gibco) at 37oC with 8% CO2. For protein expression, the baculovirus was added at 1/10 (v/v) to the culture medium of HEK293S GnTI- cells, at a density of 3 × 106 cells/mL. After 16-18 hrs, 5 mM valproic acid was added, and the cells were further incubated at 30oC with 8% CO2 for 48 hrs. The cells were collected by centrifugation (5,000 × g, 12 min, 4oC) and lysed by sonication in buffer, containing 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, and protease inhibitors (1.7 μg/mL aprotinin, 0.6 μg/mL leupeptin, 0.5 μg/mL pepstatin and 1 mM PMSF). After cell debris removal by centrifugation (4,000 × g, 12 min, 4°C), the membrane fraction was collected by ultracentrifugation (186,000 × g, 1 hr, 4°C). The membrane fraction was solubilized in buffer, containing 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 10% glycerol, 2 mM 2-mercaptoethanol (β-ME), and 1% digitonin (Calbiochem), for 1 hr at 4°C. After ultracentrifugation (186,000 × g, 20 min, 4°C), the supernatant was collected and incubated with anti-FLAG M2 affinity gel (Sigma) for 1 hr at 4°C. The resin was washed with 10 column volumes of wash buffer, containing 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 20% glycerol, 2 mM β-ME, and 0.1% digitonin. Bound prestin was eluted with the wash buffer containing 0.125 mg/mL of FLAG peptide, and EGFP fluorescence positive elution fractions were collected. For further purification, CNBr-Activated Sepharose 4 Fast Flow Beads (GE Healthcare) conjugated with anti-GFP nanobodies 44 were added to the collected fractions and incubated for 1 hr at 4°C. The resin was washed with 10 column volumes of the same wash buffer and then gently suspended with TEV protease (purified in-house) overnight at 4°C to cleave the C-terminal FLAG-EGFP tag. After the TEV protease cleavage, the flow-through fraction was pooled, concentrated to 5-10 mg/mL using a centrifugal filter device (Millipore 100 kDa MW cutoff) and loaded onto a Superose 6 Increase 10/300 GL column (GE Healthcare), equilibrated in buffer containing 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 2 mM β-ME, and 0.1% digitonin. The peak fractions were pooled and concentrated to 5-10 mg/mL.
Electron microscopy sample preparation
After precipitating aggregated proteins by ultracentrifugation at 138,000 × g for 20 min, a 3 μL portion of the supernatant was spotted onto a glow-discharged holey carbon grid (Quantifoil R1.2/1.3, Cu/Rh, 300 mesh), which was plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI) at 6˚C with a blotting time of 4 sec with 100% humidity. To obtain sulfate- and salicylate-bound hPresTS, the purified protein solution was mixed with sodium sulfate (10 mM) or sodium salicylate (30 mM), respectively, and incubated on ice for 15 min before grid preparation.
Electron microscopy data collection and processing
The prepared grids were transferred to a Titan Krios G4 microscope (Thermo Fischer Scientific), equipped with a Gatan Quantum-LS Energy Filter (GIF) and a Gatan K3 Summit direct electron detector. The camera was operated in the correlated double sampling (CDS) mode. The dataset was collected at a nominal magnification of ×105,000, corresponding to a calibrated pixel size of 0.83 Å per pixel (The University of Tokyo, Japan). Each movie was recorded for 5.0 seconds and subdivided into 64 frames. The electron flux rate was set to 7.5 e− /pix/s at the detector, resulting in an accumulated exposure of 54 e−/Å2 at the specimen. The data were automatically acquired using the SerialEM software 45, with a defocus range of -0.8 to -1.6 μm.
Initially, all datasets were corrected for beam-induced motion, using the motion correction program implemented in the single-particle analysis software RELION-3.1 46, and the contrast transfer function (CTF) parameters were estimated using CTFFIND4 47. For the dataset of the chloride-bound state, 1,373,022 particles were picked from 4,680 micrographs by using the Laplacian-of-Gaussian picking function in RELION-3.1, and were used to generate two-dimensional (2D) models for reference-based particle picking. Particles were extracted with down-sampling to a pixel size of 3.63 Å/pix and subjected to several rounds of 2D and 3D classifications. The best class contained 341,744 particles, which were then re-extracted with a pixel size of 1.10 Å/pix and subjected to 3D refinement. The resulting 3D model and particle set were subjected to per-particle CTF refinement, beam-tilt refinement, Bayesian polishing 48, and 3D refinement. The final 3D refinement and postprocessing of the three classes yielded maps with global resolutions of 3.52 Å, according to the FSC = 0.143 criterion 49. The local resolution was estimated using RELION-3.1. The processing strategy is described in Supplementary Fig. 3. For the dataset of the sulfate-bound state, 1,181,278 particles were picked from 4,077 micrographs, and the best class containing 249,144 particles was selected and processed as above, yielding a map with a global resolution of 3.52 Å (Supplementary Fig. 4). For the dataset of the salicylate-bound state, 848,704 particles were picked from the 3,375 micrographs by reference-based particle picking with the map of the chloride-bound state. The best class containing 113,410 particles was selected and processed, yielding a map with a global resolution of 3.57 Å (Supplementary Fig. 5).
Model building and validation
The models of the chloride-bound state of hPresTS were manually built de novo in the Cryo-EM density map in COOT 50, facilitated by the previously reported crystal structures of SLC26Dg (PDB ID: 5DA0). Structure refinement was initially performed with Rosetta 51 and phenix.real_space_refine 52,53. After manual adjustments, the models were then subjected to structure refinement with the Servalcat pipeline using REFMAC5 54,55 and manual real-space refinement in COOT 50. The models of the salicylate-bound state were built by using the chloride-bound model as the starting model. The statistics of the 3D reconstruction and model refinement are summarized in Supplementary Data Table 1. All molecular graphics figures were prepared with CueMol (http://www.cuemol.org).
NLC measurement
HEK293T-based stable cell lines expressing HgPres-mTq2 constructs in a doxycycline-dependent manner were established and maintained in DMEM, supplemented with 10% FBS and 1 µg/ml puromycin (Fisher Scientific), as previously described 56,57. The expression of the HgPres constructs was induced by adding 1 µg/mL doxycycline to the culture medium, one day prior to NLC recording. Whole-cell NLC recordings were performed at room temperature, using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). Recording pipettes were pulled from borosilicate glass to achieve initial bath resistances averaging 3-4 MΩ. Recordings were performed using sinusoidal (2.5-Hz, 120-150 mV amplitude) voltage stimuli superimposed with two sinusoidal stimuli (390.6 (f1) and 781.3 (f2) Hz, 10 mV amplitude). Recording pipettes were filled with an intracellular solution containing 140 mM CsCl, 2 mM MgCl2, 10 mM EGTA, and 10 mM HEPES (pH 7.3). Cells were bathed in an extracellular solution containing 120 mM NaCl, 20 mM TEA-Cl, 2 mM CoCl2, 2 mM MgCl2, and 10 mM HEPES (pH 7.3). Osmolality was adjusted to 304 mOsmol/kg with glucose. Current data were collected and analyzed by jClamp (SciSoft Company, New Haven, CT) 58.
NLC (Cm) data were analyzed using the following equation:
![](data:image/png;base64,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)
where α is the slope factor, Qmax is the maximum charge transfer, Vm is the membrane potential, Vpk is the voltage at which the maximum charge movement is attained, and Clin is the linear capacitance. The magnitude of NLC (Cm - Clin) was corrected for cell size (Clin) because larger cells tend to express greater amounts of the prestin protein (Csp ≡ (Cm - Clin)/Clin).
Cell surface protein labeling and quantitation
Stable cells were seeded on a 6-well plate, and the expression of mTq2-tagged HgPres constructs was induced by 3 µg/mL doxycycline for 2 days prior to labeling. Cells were washed once with PBS, and 2 mL of 10 µM Sulfo-Cyanine3 NHS ester (Lumiprobe) dissolved in ice-cold PBS was added (per well) and incubated for 30 minutes at 4 ºC. The reaction was stopped by the addition of 200 µL of 100 mM glycine. Cells were collected and lysed by sonication on ice in 500 µL of lysis buffer (150 mM NaCl, 20 mM HEPES, pH 7.5, 1 mM EDTA, 20 mM DDM, 1 mM DTT, and 50 µg/mL leupeptin). The lysate was centrifuged at 16,000 × g for 5 minutes at 4ºC. A GFP selector slurry (5 µL, NanoTag Biotechnologies) was added to the supernatant and incubated for 30 minutes at 4ºC, with end-over-end mixing using a rotator. Bound proteins were collected alongside with the GFP selector by brief centrifugation, and observed with a fluorescent microscope (Leica DMIRB) controlled by µManager 59. Merged images of GFP selectors in cyan and red channels were analyzed using FIJI 60 to determine the fluorescent signal intensities of mTq2 and Cy3. The results are shown in Supplementary Fig. 7.
Molecular dynamics simulation
We performed molecular dynamics (MD) simulations to analyze interactions of a chloride ion (Cl-) binding to prestin. Wild-type hPres model was constructed by modeller 61 , using the chloride-bound hPresTS cryo-EM structure as the template. The system includes residues from Arg58 to Pro506. Hydrogen atoms were added using the psfgen plugin in NAMD. The protonation states of ionizable residues were assumed to be those at pH 7 by PROPKA. The structure of prestin was manually embedded in a 105 Å × 105 Å 1-palmitoy-2-oleoyl-sn-glyceroo-3-phosphocholine (POPC) bilayer modeled with VMD. The bilayer was solvated with TIP3P water molecules with Na+ and Cl- ions of 0.15 M using the solvate plugin and the autoionize plugin in VMD. The simulation box was initially 105 Å × 105 Å × 120 Å in size and contained 110,295 atoms.
We first performed a preparatory MD simulation with the NAMD program package. The force field parameters of the protein, water molecules, and lipids were CHARMM27, TIP3P, and CHARMM36, respectively. Short-range non-bonded interactions were cutoff at 12 Å with a force-switching function. Long-range electrostatic interactions under periodic boundary conditions were calculated with the particle mesh Ewald method. Temperature and pressure were controlled with Langevin dynamics and the Langevin piston method, respectively. In regulation of the pressure, the x and y axes were isotropically scaled (NPTiso), where the z axis was perpendicular to the membrane surface. The initial degrees of freedom of water molecules were constrained with the SETTLE algorithm. Bond including hydrogen atom were constrained with the RATTLE method. First, a 1,000-step energy minimization under the NVT condition were performed. Then, a 160-ps heating to 200 K under the NVT condition and a 40-ps heating simulation to 300 K under the NPTiso condition with the protein, water molecules, and lipid tails only being allowed to move. The time step of trajectory calculation was set to 2 fs. After 10-ns equilibrium MD simulation under NPTiso condition with the water molecules, and lipids tails only being allowed to move, 10-ns equilibrium MD simulation under NPTiso condition without any restraint was performed. The force field parameters were then switched to those of the Amber force field (ff14SB, lipid17, and TIP3P for the protein, lipids, and water molecules, respectively), and a 50-ns equilibrium simulation was carried out with NAMD. Finally, from the last snapshot of the equilibrated MD system obtained by the preparatory simulation described above, a sampling MD simulation for 100 ns was performed with pmemd.cuda.MPI of the AMBER16 program package with the Amber force fields (ff14SB, lipid14, and TIP3P for the protein, lipids, and water molecules, respectively).
A stable binding of the Cl- ion in the binding pocket was observed during the MD simulation for 100 ns (Supplementary Fig. 8). It is noteworthy that the binding of the Cl- ion in the binding pocket was not achieved by tight binding with the protein side-chains in the binding pocket such as Arg399, Ser396, and Ser398. The Cl- ion was not directly attached to Arg399 (Supplementary Fig. 8b), and the hydroxy groups of Ser396 and Ser398 frequently detached from the Cl- ion (Supplementary Fig. 8c). The stable accommodation of the Cl- ion in the binding pocket without the strict recognition of the Cl- ion by tight binding of the protein groups implies that the Cl- ion binding is mainly fulfilled by a strongly positive electrostatic environment at the binding pocket created by a positive charge of Arg399 and helix dipoles of the symmetrically arranged TM3 and TM10, rather than by precise molecular recognition.
Data availability
Cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-31757 (chloride-bound), 31758 (sulfate-bound) and 31759 (salicylate-bound). Atomic coordinates have been deposited in the Protein Data Bank under IDs 7V73 (chloride-bound), 7V74 (sulfate-bound) and 7V75 (salicylate-bound). The raw images have been deposited in the Electron Microscopy Public Image Archive, under accession code EMPIAR-xxxxx.