Abstract
The voltage-dependent motor protein prestin (also known as SLC26A5) is responsible for the electromotive behaviour of outer-hair cells and underlies the cochlear amplifier1. Knockout or impairment of prestin causes severe hearing loss2,3,4,5. Despite the key role of prestin in hearing, the mechanism by which mammalian prestin senses voltage and transduces it into cellular-scale movements (electromotility) is poorly understood. Here we determined the structure of dolphin prestin in six distinct states using single-particle cryo-electron microscopy. Our structural and functional data suggest that prestin adopts a unique and complex set of states, tunable by the identity of bound anions (Cl− or SO42−). Salicylate, a drug that can cause reversible hearing loss, competes for the anion-binding site of prestin, and inhibits its function by immobilizing prestin in a new conformation. Our data suggest that the bound anion together with its coordinating charged residues and helical dipole act as a dynamic voltage sensor. An analysis of all of the anion-dependent conformations reveals how structural rearrangements in the voltage sensor are coupled to conformational transitions at the protein–membrane interface, suggesting a previously undescribed mechanism of area expansion. Visualization of the electromotility cycle of prestin distinguishes the protein from the closely related SLC26 anion transporters, highlighting the basis for evolutionary specialization of the mammalian cochlear amplifier at a high resolution.
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Data availability
The atomic structure coordinates have been deposited at the RCSB PDB under accession numbers 7S8X, 7S9A, 7S9B, 7S9C, 7S9D and 7S9E; and the EM maps have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-24928, EMD-24930, EMD-24931, EMD-24932, EMD-24933 and EMD-24934. All materials generated during the current study are available from the corresponding author under a materials transfer agreement with The University of Chicago.
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Acknowledgements
We thank M. Zhao for discussions and assistance with imaging; K. Poole, C. Cox, T. Ngo, O. Bavi, R. Hulse, C. Bassetto, Y. Nikolaev, Y. Krishnan, P. Bezanilla, H. Mchaourab, P. Gueorguieva, S. Zhong, M. Karami, P. Haller and F. Galan, and the members of the Perozo laboratory for exchanging ideas and comments on the manuscript; P. Shi for sharing the Tursiops prestin plasmid; J. Fuller, J. Austin II and T. Lavoie at the University of Chicago Advanced Electron Microscopy Facility for microscope maintenance and training; and U. Baxa and T. J. Edwards at NCEF for cryo-EM data collection. Anton 2 computer time was provided by the Pittsburgh Supercomputing Center (PSC) through grant R01GM116961 from the National Institutes of Health. The Anton 2 machine at PSC was made available by D.E. Shaw Research. N.B. acknowledges the Biology of Inner Ear course (BIE2019) and Gordon Conference (Auditory System Gordon Research Conference) for inspiring him to study hearing and prestin. This work was supported by NIDCD grant R01 DC019833 to E.P. N.B. was the recipient of a Chicago Fellowship. M.D.C. was supported by F30MH116647 and T32GM007281. This research was in part supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E.
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N.B., M.D.C. and E.P. conceived the project. N.B. expressed and purified the protein. N.B. and M.D.C. prepared cryo-grids. N.B. and B.G.R. performed EM data collection. N.B., B.G.R. and M.D.C. processed the cryo-EM data and built and refined the atomic models. N.B. and G.F.C. performed and analysed the electrophysiological experiments. R.S. carried out MD simulations and electrostatic calculations. N.B. and W.M. carried out molecular cloning, mutagenesis and created all of the expression constructs. N.B. and W.M. managed cell culture. All of the authors analysed the data. N.B. and E.P. wrote the manuscript with input from all of the other authors.
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Extended data figures and tables
Extended Data Fig. 1 Function, biochemistry and structural features of electromotile prestin.
a, Electromotility analysis of HEK293 cells transfected with wild-type dolphin prestin compared to GFP-only transfected cell (Mock-GFP). The cellular displacement was normalized based on the cell largest diameter, d0 (Fig. 1b). The normalized electromotility was 0.05±02 (n = 6) versus 0.008±0.002 (n = 5) for wild-type prestin and Mock-GFP, respectively. These values were measured at the depolarizing voltage step changing from +120 mV to −120 mV (mean ± SD, is the number of independent cells. One-sided Student t-test, unpaired, P=0.005). b, Size-exclusion chromatography (SEC) curves of the full-length dolphin prestin purified in GDN, run on a Superose 6 column, in high Cl− (red) and SO42− (blue) based solution. The fractions indicated by black dotted lines in both represent purified proteins that were used for cryo-EM imaging. c, Purified dolphin prestin cryo-EM samples, run on a Stain-free SDS-PAGE gel, indicating size of ~80 kDa for the full-length prestin monomer (representation of n = 3).d, Topology of dolphin prestin. Different domains are indicated by color; the gate domain is colored in blue, the core domain in red and the C- and N-termini as well as the STAS domain in grey. The transmembrane helices are numbered from 1 to 14. The N- and C-termini as well as the STAS domain are oriented towards the cytoplasm.
Extended Data Fig. 2 Flow chart for the cryo-EM data processing and structure determination of the dolphin prestin in high Cl− condition.
a, The final reconstruction has a nominal resolution of 3.3 Å (at FSC=0.143). The yellow scale bar on the micrograph represents 200 Å. All the images in this figure were created in UCSF ChimeraX.
Extended Data Fig. 3 Structure of prestin in high Cl− and comparison with the Intermediate.
a, Comparison between prestin (high Cl−) (blue and Red) and SLC26A9 Intermediate state (6RTF, grey). The structures are aligned based on residues 460 to 505 of one subunit (TM13-TM14, dotted box). ChimeraX was used for illustration. b, Electrostatic potential and surface charge distribution of SLC26A9 intermediate state19 compared with that of prestin in high Cl− panel c. The electrostatic charge distribution ranges from −5 to 5 kT from negative to positive charge. ChimeraX was used for illustration.
Extended Data Fig. 4 Flow chart for the cryo-EM data processing and structure determination of the dolphin prestin in SO42−.
a, b Cryo-EM data processing and structure determination of the dolphin prestin in Down I (SO42−) and Down II (SO42−) states. A was obtained from Dataset I, which was combined with Class B from Dataset II. The final reconstruction yielded two structures, Down I (SO42−) and Down II (SO42−), which have nominal resolutions of 4.2 and 6.7 Å, respectively (at FSC=0.143). See Supplementary Figure 5 for the steps on how Class A and B were further processed. Evidence of both states was found in dataset II, however merging of datasets was required to improve resolution of states. c, Flow chart for the cryo-EM data processing and structure determination of the dolphin prestin in the Intermediate state (SO42−) (See Methods for details). The final reconstruction has a nominal resolution of 4.6 Å (at FSC=0.143). UCSF ChimeraX was for illustration of all the structures. The yellow scale bar on all the micrographs represents 200 Å.
Extended Data Fig. 5 Prestin’s cross-sectional area changes upon transition from Down to Up states.
a, Upon the transition from Down to Up state and the movement of the anion-binding site, the most obvious changes are seen in the peripheral helices TM5b, TM6-TM7, and TM8. b, MD simulation of prestin in Up state is compared with the Inhibited II state (Cl− and Salicylate) equilibrated in POPC lipid bilayers. The cross-sectional area of outer and inner monolayers with mapped leaflet coordinate in the Z direction (across the membrane thickness) using all-atom molecular dynamics simulations (1µs). Δz shows movement of the phosphate group of the lipids in the Z (thickness) direction. The comparison was made between Up (Cl−) and Inhibited II (SO42−) states. The largest difference was observed at the location of the TM6 helix. c, Cross-sectional area calculations of the transmembrane domain of SLC26A9(12) along the hydrophobic thickness using CHARMM-membrane builder. Cross-sectional area change of SLC26A9 from Inward-facing to Intermediate states (6RTC and 6RTF) per monomer19. Note that prior to area calculation, the spatial arrangements of all the structures with respect to the hydrocarbon core of the lipid bilayer were first adjusted using the PPM server(30). The structures were aligned based on residues 460 to 505 (TM13-TM14). d, Comparison of the change in the micelle morphology between two salicylate-inhibited structures Inhibited I (Cl−) and Inhibited II (SO42− + Salicylate) states. The overlay of the two states shows drastic changes in the micelle thickness especially around TM6 region in addition to the overall changes in the micelle in-lane direction, both indicative of major structural rearrangements between the two states. ChimeraX was used for illustration.
Extended Data Fig. 6 Salicylate outcompetes SO42− in binding to anion-binding pocket.
a, The NLC measurements of HEK293T cells transfected with dolphin prestin in SO42− (0.15±0.06; n = 6). The NLC of these cells were completely abrogated (0.01±0.01) by 10 mM Na-Salicylate (mean ± s.e.m.; n, is the number of independent cells. One-sided student’s t-test, unpaired, P=0.01) b, Density of Salicylate (orange) in the anion-binding site (blue) was resolved in the Inhibited II (SO42−) state of dolphin prestin. c, Sequence alignment of prestin and close SLC transporters across different species. Residues forming the anion-binding site are largely conserved (e.g. Q97, F101, F137). Putative voltage-sensing residue R399 in dolphin prestin is replaced by a valine in murine SLC26A9. Clustal Omega was used for the sequence alignments. ChimeraX was used for illustration.
Extended Data Fig. 7 Flow chart for the cryo-EM data processing and structure determination of the dolphin prestin in the Salicylate-Inhibited states.
Flow chart of the dolphin prestin in the a, Inhibited I state (Cl− + Salicylate) and b, (SO42− + Salicylate) The final reconstructions have a nominal resolution of 3.8 Å and 3.7 Å, respectively (at FSC=0.143). All the images in this figure were created in UCSF ChimeraX. The yellow scale bar on all the micrographs represents 200 Å.
Extended Data Fig. 8 Electrostatic calculations and charge transfer of prestin across the membrane.
a, Mutation of the key residues in the anion binding pocket either completely abolishes the NLC (R399Q) or right shifts the V1/2 by more than 80 mV (F101Y) to around +25 ± 5 mV (mean ± s.e.m.; n, is the number of independent cells. One-sided Student t-test, P=0.001); a similar effect has been observed in other prestin homologues using patch-clamp electrophysiology (51). b, Snapshots from the MD trajectories of the systems, and calculation of the electrostatic potential across the membrane at two states, the Down I state (with SO42− in the left cavity, and without SO42− in the right cavity) versus Up (with Cl− in the left cavity and without any Cl− in the right cavity). The x-z plane is crossing the two central anion-binding sites. In both models, the positive field is mainly focused around the transmembrane mid-plane and around the anion-binding site, creating an attractive (blue) field for the binding of the anion. However, in the Up state the field is more positive around the mid-plane compared to the corresponding region in the Intermediate state. In both cases, the presence of the anion only partially neutralizes (~35%) the positive field around the bilayer mid-plane. Note that the actual size of the simulation box is larger than what is illustrated here (see Methods). c, Averaged 1-D fraction of membrane potential in the z direction along the two central binding sites (shown as dashed blue lines in panel A with the central binding sites highlighted using the red cross symbols). The 1-D and 2-D maps were directly extracted from the ensemble averaged 3-D fraction of membrane potential map. The location of the phosphate atoms of the outer and inner lipid leaflets along the z axis was highlighted with dashed gray lines). d, Displacement of charge for prestin in the Up and Down I conformations at different transmembrane potentials. The gating charge between the two states is 0.38 +0.25 e calculated as the offset constant between the linear fits. (n = 3; data are mean ± SD; One-sided Student’s t-test; P=0.05). e, R399 in both monomers have been mutated to Q, S and E in different systems to see the contribution of R399 residue to the positive charge at the bilayer mid-plane using electrostatic calculations. R399 mutation to polar residues shows that R399 has almost ~40% contribution the positive charge of the field at the bilayer mid-plane. The remainder likely comes from the TM3-TM10 helical dipole and other positive charges in this area.
Extended Data Fig. 9 Whole cell patch-clamp electrophysiology of the mutations of different glycine residues along the TM6 helix.
All the individual data points, that has been averaged in Fig. 3f, has been presented here. Compared to wild-type prestin, mutation of evolutionary conserved glycine residues, a, G274 and G275 and b, G263, G265 and G270 largely affects the NLC.
Supplementary information
Supplementary Information
This file contains further supportive results for the findings in this study, including Supplementary Figs. 1–10 (which include the uncropped gel source data).
Supplementary Video 1
Electromotility measurements of HEK293 cells transfected with dolphin prestin using whole-cell patch-clamp electrophysiology. To evoke prestin-mediated electromotility, the membrane potential was held at −70 mV; 10 mV increase-in-amplitude voltage steps were applied up to the final steps, which was from +150 mV to −140 mV (Fig. 1b). The magenta square indicates the area that was chosen in our custom-written code to track the cellular displacements.
Supplementary Video 2
Structural changes from the expanded (down I) to the compact (up) conformation as a linear interpolation. The side front and top views of the dimer have been shown in one single frame. The anion-binding site is highlighted in red and Arg399 is shown in stick representation and the backbone has been coloured yellow. The videos were made in UCSF ChimeraX.
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Bavi, N., Clark, M.D., Contreras, G.F. et al. The conformational cycle of prestin underlies outer-hair cell electromotility. Nature 600, 553–558 (2021). https://doi.org/10.1038/s41586-021-04152-4
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DOI: https://doi.org/10.1038/s41586-021-04152-4
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