Structures of the otopetrin proton channels Otop1 and Otop3

Abstract

Otopetrins (Otop1–Otop3) comprise one of two known eukaryotic proton-selective channel families. Otop1 is required for otoconia formation and a candidate mammalian sour taste receptor. Here we report cryo-EM structures of zebrafish Otop1 and chicken Otop3 in lipid nanodiscs. The structures reveal a dimeric architecture, with each subunit forming 12 transmembrane helices divided into structurally similar amino (N) and carboxy (C) domains. Cholesterol-like molecules occupy various sites in Otop1 and Otop3 and occlude a central tunnel. In molecular dynamics simulations, hydrophilic vestibules formed by the N and C domains and in the intrasubunit interface between N and C domains form conduits for water entry into the membrane core, suggesting three potential proton conduction pathways. By mutagenesis, we tested the roles of charged residues in each putative permeation pathway. Our results provide a structural basis for understanding selective proton permeation and gating of this conserved family of proton channels.

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Fig. 1: Proton channel function and structures of Otop1 and Otop3 homodimers in lipid nanodiscs.
Fig. 2: Lipid binding in Otop1 and Otop3.
Fig. 3: N and C domain vestibules in Otop1 and Otop3.
Fig. 4: Molecular dynamics simulations reveal hydration of potential proton pathways in Otop1.
Fig. 5: Mutation of charged residues at intrasubunit interface results in loss of function.

Data availability

Cryo-EM maps and atomic coordinates have been deposited at the Electron Microscopy Data Bank and PDB, respectively, with accession codes EMD-9360 and PDB 6NF4 (zebrafish Otop1) and EMD-9361 and PDB 6NF6 (chicken Otop3). All other data are available upon reasonable request to the corresponding authors.

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Acknowledgements

We thank W. Anderson for managing the electron microscopy facility at Scripps Research, H. Turner and J. Torres for help with data collection, C. Bowman for assistance with computation, D. Artiga for help with generating cDNA constructs and V. Katritch for providing expert advice on the design of expression constructs. We acknowledge members of the Ward, Liman, Sansom and Patapoutian laboratories for helpful advice. This work was supported by a Ray Thomas Edwards Foundation grant (to A.B.W.); funding from the NIH (No. NIDCD013741 to E.R.L.); and Wellcome (grant No. 208361/Z/17/Z), BBSRC (grant Nos. BB/N000145/1 and BB/R00126X/1) and EPSRC (grant No. EP/R004722/1; to M.S.P.S). K.S. is a postdoctoral fellow of the Jane Coffin Childs Memorial Fund for Medical Research. C.C.A.T. is supported by the Skaggs-Oxford Scholarship and the Croucher Foundation.

Author information

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Authors

Contributions

K.S. prepared cryo-EM samples, collected and processed cryo-EM data and built structures. B.T. performed electrophysiology experiments and analyzed data. C.C.A.T. performed molecular dynamics simulation and analyzed data. W.-H.L. generated constructs and conducted FSEC experiments. Y.-H.T. designed and generated constructs and analyzed data. J.P.K. generated constructs and performed confocal imaging. M.S.P.S., E.R.L. and A.B.W. supervised molecular dynamics, functional experiments and cryo-EM structure determination, respectively. K.S. drafted a majority of the manuscript, with significant additions from B.T., C.C.A.T., M.S.P.S., E.R.L. and A.B.W. All authors contributed equally to finalization of the manuscript.

Corresponding authors

Correspondence to Emily R. Liman or Andrew B. Ward.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Topology and sequence alignment of Otopetrin subtypes.

zfOtop1 and chOtop3 were aligned with mouse Otop1 (mOTOP1, Uniprot ID Q80VM9), human Otop1 (hOTOP1, Uniprot ID Q7RTM1), mouse Otop2 (mOTOP2, Uniprot ID Q80SX5), human Otop2 (hOTOP2, Uniprot ID Q7RTS6), mouse Otop3 (mOTOP3, Uniprot ID Q80UF9), human Otop3 (hOTOP3, Uniprot ID Q7RTS5). The transmembrane helices in the zfOtop1 structure are depicted as cylinders above the sequence. Linkers that were modeled in the zfOtop1 structure are depicted as solid lines, while portions of the sequence excluded from the model due to poor density are depicted as dotted lines.

Supplementary Figure 2 Proton channel properties of zfOtop1 and chOtop3.

Current magnitude as a function of pH in HEK-293 cells expressing zfOtop1 (red, n = 6), chOtop3 (cyan, n = 4) and in untransfected control cells (black triangles, n = 4). Error bars show s.e.m.

Supplementary Figure 3 Cryo-EM sample and data processing for Otop1.

FSEC traces of HEK cells expressing N-terminal GFP fusion of Otop1 solubilized in buffer containing 1% DDM (blue) or 1% DDM and 0.15% CHS (red). b, preparative SEC trace of Otop1 reconstituted in MSP2N2 nanodisc containing soybean lipids. c, SDS-PAGE analysis of purified Otop1 reconstituted in nanodiscs that was used for cryo-EM. Black arrow points to band corresponding to Otop1, while the red arrow points to band corresponding to MSP2N2 scaffold. d, representative aligned and dose-weighted cryo-EM micrograph. Scale bar = 100 nM. e, cryo-EM data processing scheme to obtain final reconstruction. f, relative angular distribution of final reconstruction used for model building. Red bars represent views with more particles. g, FSC plots of unmasked (blue) and masked (red) cryo-EM reconstructions, and model vs map comparison calculated in Phenix 1.14. (orange). h, side view (left) and centrally sliced side view (right) of Otop1 cryo-EM map sharpened to a b-factor of -80 Å2 and colored according to local resolution determined by blocres program in RELION 3.0. Color key shows local resolution in Å.

Supplementary Figure 4 Cryo-EM sample and data processing for Otop3.

a, FSEC traces of HEK cells expressing N-terminal GFP fusion of Otop3 solubilized in buffer containing 1% DDM (blue) or 1% DDM and 0.15% CHS (red). b, preparative SEC trace of Otop1 reconstituted in MSP2N2 nanodisc containing soybean lipids. c, representative aligned and dose-weighted cryo-EM micrograph. Scale bar = 100 nM. d, cryo-EM data processing scheme to obtain final reconstruction. e, relative angular distribution of final reconstruction used for model building. Red bars represent views with more particles. f, FSC plots of unmasked (blue) and masked (red) reconstructions, and model vs map comparison (orange). g, side view (left) and centrally sliced side view (right) of Otop3 cryo-EM map sharpened to a b-factor of -80 Å2 and colored according to local resolution determined by blocres program in RELION 3.0. Color key shows local resolution in Å.

Supplementary Figure 5 Fit of molecular model to cryo-EM density of Otop1 and Otop3.

a, side and top views of unsharpened maps of Otop1 (left) and Otop3 (right). Density within 2 Å of the molecular model is colored yellow (Otop1) or cyan (Otop3). Putative density for the MSP2N2 scaffold protein can be observed (gray density). b, isolated atomic model fragments of Otop1 (yellow, left panels) and Otop3 (cyan, right panels) with sharpened maps of Otop1 (blue mesh, 5 σ, left) or Otop3 (gray mesh, 4.5 σ, right panels) superimposed. c, Side (left) and top (right) views of molecular model of the Otop1 dimer, with bound CHS or cholesterol molecules depicted as sticks and numbered. d, sharpened cryo-EM maps of Otop1 superimposed onto CHS or cholesterols shown in C. σ values for the maps generated in Pymol are as follows: #1, 4 σ, #2 = 2.5 σ, #3 = 3 σ, #4 = 3 σ. e, side view of Otop3 dimer model with modeled CHS molecules shown as yellow sticks. f, Sharpened map of Otop3 at 3 σ is superimposed onto the CHS molecule. g, unsharpened cryo-EM map of Otop1 low-pass filtered to 4 Å resolution reveals weak density for the TM6-TM7 linker (highlighted by dashed yellow line), demonstrating the connectivity of the N and C domains within a single subunit. 18 residues are missing from the atomic model in the TM6-TM7 linker, which is sufficient to span this distance as an extended coil.

Supplementary Figure 6 Structural conservation of Otopetrins, and comparison of Otop1 to LacY, a canonical MFS transporter, and Acrb, a canonical RND protein.

a, Structural alignments of Otop1 (blue) and Otop3 (red) based on the entire dimer, a single subunit, N domain, or C domain. Alignments and RMSD values were calculated using the ‘align’ program in Pymol. b-d, side (b,c) and top (d) views of the Otop1 dimer, colored by Consurf sequence conservation score. Magenta represents more conserved, and teal represents less conserved. Bound cholesterol or CHS molecules are depicted as yellow sticks. In c, subunit 1 is shown as cartoon and subunit 2 is shown as ribbon representation to help differentiate the subunits. e-g, Side and top views of the Otop1 subunit (e), LacY lactose permease (f, PDB ID 1PV6), and Acrb multidrug efflux transporter (g, PDB ID 1IWG). Like Otopetrins, LacY and Acrb have twelve TM helices split into two 6-TM helix-containing bundles, the N (green) and C (light green) domains related by a two-fold pseudosymmetry axis (red ellipsoids). The TM helices are numbered to show that despite the similarity in domain organization, the folds of Otop1, LacY, and Acrb are distinct.

Supplementary Figure 7 Intersubunit and intrasubunit interfaces.

a-d, representations of the intrasubunit interface in Otop1 (a) and Otop3 (c). b,d show expanded views of the interface with interfacial side chains shown in stick representation and labeled. The bound cholesterol molecule at the intrasubunit interface in Otop1 is shown. e, model of Otop1, with one subunit colored purple and the other subunit colored green. f, expanded view of boxed region in e, with residues contributing to intersubunit interface shown as stick. g, h, overall (g) and expanded (h) views of Otop3 intersubunit interface. i, FSEC analysis of N-terminally GFP-fused wild type zebrafish Otop1 and constructs harboring alanine mutations at two tryptophans (W394A, W398A, bolded in f) at the intersubunit interface. The mutants display right-shifted peaks compared to the wild type dimeric peak, consistent with solubilization of monomeric species. j, current magnitude at pH 6.0–5.0 in HEK-293 cells expressing N-terminally GFP fused zfOtop1 (gray, n = 5), or its mutants W394A (red, n = 5) and W398A (blue, n = 4). Wild type controls are duplicated from Supplementary Fig. 1. Error bars show s.e.m. k-m, confocal images (left panels) showing HEK-293 cells expressing tag-RFP and the Otop1 constructs in i,j. Each image is at the same scale, and scale bar (5 µm) is shown in k. Right panels show fluorescence intensity for GFP (green trace) and RFP (red trace) channels along the yellow line in the images. Surface expression, as evident by peak GFP expression outside the region of peak RFP expression, can be observed for wildtype zfOtop1, but not for the mutants. Note that the GFP fused channels are excluded from the nucleus (not labelled) which occupies much of the cell.

Supplementary Figure 8 Dynamic arrangements of cholesterol molecules associated with Otop1 and Otop3 in MD simulations and their role in protein stabilization.

a, for Otop1, the relative positions of the six cholesterol molecules occupying the central tunnel were well maintained throughout the 100-ns simulation. In contrast, the cholesterol molecule occupying the intrasubunit interface has a greater mobility. b, when six cholesterol molecules were placed in the central tunnel of Otop3, some vertical displacements and rotations were seen which could be attributed to its wider central tunnel. c, distances between the center of geometry (COG) of Otop1 and the COGs of cholesterol molecules initially associated with the intrasubunit interface, plotted against simulation time (colored lines). Only in one of the six instances (Run 3, Subunit 1) the cholesterol molecule remained sandwiched between N and C domains throughout the 100 ns-simulation. The corresponding distances for the cholesterol molecules in the central tunnel of all runs are plotted as black lines, reflecting their stable arrangement within the tunnel. d, similar plot as c for Otop3, note the cholesterol molecules in the central tunnel were more dynamic in nature compared to those in Otop1. e, Root-mean-square deviations (RMSDs) of the transmembrane residues’ backbone atoms for the dimer (black line) and at individual domain level (colored lines), with different membrane lipid compositions. For Otop1, conformational drift was reduced in the presence of cholesterol molecules in the central tunnel and at the intrasubunit interface. Otop3, when contrasted with Otop1, has greater RMSDs at the dimer level but not at the individual domain level, which could be due to our insertion of cholesterol molecules in the central tunnel.

Supplementary Figure 9 Conserved polar interactions in the putative Otop pores.

a, b, Top (a) and side (b) views of N domains and C domains of Otop1 and Otop3 aligned and superposed. TM1/7 are removed from a and TM1/2/7/8 are removed from b for clarity. The conserved QNY triad is shown as sticks. c, d, side view of the QNY triad in the N domains of Otop1 (c) and Otop3 (d). e, f, side view of the QNY triad in the C domains of Otop1 (e) and Otop3 (f), as well as additional interactions involving a conserved arginine and opposing glutamic acid (e) or glutamine (f). In c-f, sharpened cryo-EM maps (blue mesh, 5σ) are superimposed onto the models, and interatomic distances highlighting potential interactions are depicted with dashed lines and labeled with distances (in Å).

Supplementary Figure 10 Hydration of potential proton pathways in Otop3 revealed by MD simulations.

Data is presented in the same manner as in Fig. 4 (for Otop1). af, snapshots at the end of a 100-ns simulation (Run 3, see Supplementary Dataset 1; df show snapshots from subunit 1 with the equivalent side chains as Otop1 labeled). In this example the intrasubunit interface (a) remained largely dewetted in the cytoplasmic half throughout the simulation, but in other occasions (for example Run 2 Subunit 2) the entire interface was wetted throughout. Like in Otop1, the intersubunit interface (b) was not wetted at all. c, cholesterol molecules in the central tunnel exclude water passage completely although this is likely not the exact configuration natively. de, The upper wetted and lower less hydrated regions were seen, similar to Otop1. fg, A higher incidence of dehydration at the C domain and the intrasubunit interface was observed for Otop3 compared to Otop1. g, the density distributions of water oxygen atoms along the three potential proton pathways, averaged across two subunits and three simulations. Positions of important side chains are drawn as rectangles, with the bounds representing μ ± 2σ. h, Electrostatic surface potential (contoured from −5 kT (red) to 5 kT (blue)) of the same snapshot in af and same view as a, in stereo representation. The surface is clipped ~15 Å from the front to highly the potential along the putative pores. Qualitatively the electrostatic distribution is similar to that in Otop1.

Supplementary information

Supplementary Information

Supplementary Figs. 1–10

Reporting Summary

Supplementary Dataset 1

Raw data of water oxygen distribution along the three potential proton pathways, displayed as individual MD simulation run and subunit. For the N domain of Otop1 and Otop3, and the C domain of Otop1, stochastic wetting and dewetting in the cytoplasmic halves (below the QNY triad) were observed. The C domain of Otop3 remained dewetted in all repeats. The intrasubunit interface of Otop1 has a more uniform distribution than the N and C domains and in 5 of the 6 instances remain largely wetted in the simulations. Otop3 however, has a less wetted cytoplasmic half below the conserved E265. The righthanded density distributions are combined to give Fig. 4g (Otop1) and Supplementary Fig. 10 (Otop3).

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Saotome, K., Teng, B., Tsui, C.C.(. et al. Structures of the otopetrin proton channels Otop1 and Otop3. Nat Struct Mol Biol 26, 518–525 (2019). https://doi.org/10.1038/s41594-019-0235-9

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