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Structure and mechanogating of the mammalian tactile channel PIEZO2

Nature volume 573, pages 225–229 (2019)Cite this article

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Abstract

PIEZO2 is a mechanosensitive cation channel that has a key role in sensing touch, tactile pain, breathing and blood pressure. Here we describe the cryo-electron microscopy structure of mouse PIEZO2, which is a three-bladed, propeller-like trimer that comprises 114 transmembrane helices (38 per protomer). Transmembrane helices 1–36 (TM1–36) are folded into nine tandem units of four transmembrane helices each to form the unusual non-planar blades. The three blades are collectively curved into a nano-dome of 28-nm diameter and 10-nm depth, with an extracellular cap-like structure embedded in the centre and a 9-nm-long intracellular beam connecting to the central pore. TM38 and the C-terminal domain are surrounded by the anchor domain and TM37, and enclose the central pore with both transmembrane and cytoplasmic constriction sites. Structural comparison between PIEZO2 and its homologue PIEZO1 reveals that the transmembrane constriction site might act as a transmembrane gate that is controlled by the cap domain. Together, our studies provide insights into the structure and mechanogating mechanism of Piezo channels.

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Fig. 1: The homotrimeric structure of PIEZO2.
Fig. 2: The 38-transmembrane-helix topological organization.
Fig. 3: The ion-conducting pore.
Fig. 4: Coordinated displacement of the cap domain and the transmembrane gate.

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Data availability

The structural coordinates of mouse PIEZO2 have been deposited in the PDB under the accession 6KG7. The cryo-EM map was deposited into the Electron Microscopy Data Bank (EMDB) under the accession 9975.

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Acknowledgements

We thank A. Patapoutian for sharing the mouse Piezo2 cDNA and the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing) for providing cryo-EM facility support. This work was supported by grant numbers 2016YFA0500402, 31825014, 31630090 and 2015CB910102 to B.X. and 31570730, 2016YFA0501102 and 2016YFA0501902 to X.L., from either the National Key R&D Program of China or the National Natural Science Foundation of China.

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Author notes

  1. These authors contributed equally: Li Wang, Heng Zhou, Mingmin Zhang, Wenhao Liu

Authors and Affiliations

  1. State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, IDG/McGovern Institute for Brain Research, School of Pharmaceutical Sciences, Tsinghua University, Beijing, China

    Li Wang, Mingmin Zhang, Wenhao Liu, Tuan Deng, Qiancheng Zhao, Yiran Li & Bailong Xiao

  2. MOE Key Laboratory of Protein Sciences, Beijing Advanced Innovation Center for Structural Biology, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Heng Zhou, Jianlin Lei & Xueming Li

  3. Joint Graduate Program of Peking-Tsinghua-NIBS, School of Life Sciences, Tsinghua University, Beijing, China

    Mingmin Zhang, Wenhao Liu & Bailong Xiao

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  1. Li Wang
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Contributions

L.W. carried out protein purification, electron microscopy sample preparation and data collection; H.Z. performed electron microscopy sample preparation, data collection, image processing and model building; M.Z. did electrophysiology; W.L. did electrophysiology and plasmid preparation; T.D. did mutagenesis; Q.Z. cloned the expression vector and helped with initial testing of expression; Y.L. helped with experiments; L.L. assisted in collection of electron microscopy data; X.L. supervised collection and analysis of electron microscopy data; B.X. conceived and directed the study, analysed the structure, made the figures and wrote the manuscript with help from all other authors.

Corresponding authors

Correspondence to Xueming Li or Bailong Xiao.

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Peer review information Nature thanks Boris Martinac, Edwin McCleskey and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Optimization of the purification of recombinant PIEZO2 proteins.

a, Schematic of the PIEZO2-pp-GST-IRES-GFP expression construct driven by the cytomegalovirus (CMV) promoter. b, Representative traces of inward currents at −80 mV evoked by a series of mechanically probing steps in 1-μm increments in whole-cell configuration in PIEZO1-KO-HEK cells transfected with the indicated constructs. The number of cells recorded is shown in c. P2, PIEZO2.c, d, Scatter plots of the maximal poking-induced currents (Imax) (c) and the inactivation τ (d). Data are mean ± s.e.m. and the number of recorded cells is labelled. Two-tailed unpaired Student’s t-test; P = 0.2347 (c) and P = 0.5826 (d). e, Western blotting of cell lysates derived from HEK293T cells transfected with the indicated constructs. The anti-GST antibody detected the PIEZO1-GST and PIEZO2-GST proteins, and the anti-β-actin antibody detected β-actin for a loading control. m indicates a mouse-derived protein. f, Representative traces of gel filtration of purified PIEZO1 or PIEZO2 proteins using the detergent C12E10. UV, ultraviolet. g, A representative trace of gel filtration of the purified full-length PIEZO2 with the GST affinity tag cleaved, in the detergent GDN. h, Coomassie blue staining showing the purified PIEZO2 proteins separated on an 8% SDS–PAGE gel. i, Representative negative-staining electron microscopy images of PIEZO2 purified in either C12E10 or GDN. The experiments in e–i were independently repeated at least three times with similar results.

Extended Data Fig. 2 Determination of the cryo-EM structure of PIEZO2.

a, Representative cryo-electron micrograph of PIEZO2 solubilized in the detergent GDN. b, Power spectrum of the micrograph in a, with the 3.0 Å frequency indicated. c, Gold-standard Fourier shell correlation (FSC) curves of the indicated density maps. Reported resolutions were based on the FSC = 0.143 criterium. d, Representative 2D class averages of PIEZO2 particles, showing the three-bladed, propeller-like top view and a bowl-like side view. e–h, Top, local-resolution maps of the indicated densities. Bottom, Euler angle distribution of particles that were used in the final 3D reconstruction; the height of the cylinder is proportional to the number of particles for that view.

Extended Data Fig. 3 Flowchart of electron-microscopy data processing.

Details of data processing are described in the ‘Imaging processing’ section of the Methods.

Extended Data Fig. 4 Local electron-microscopy density of the indicated domains of PIEZO2.

The helices are shown in cartoon representation with side chains as sticks. The cryo-EM density is shown as grey mesh. Clearly resolved residues with bulky side chains—for example, phenylalanine, tryptophan, tyrosine, proline and histidine—are labelled. The three pore-lining inner helices are shown in both side and top views to highlight the side-chain density of F2754, which forms a transmembrane gate, and E2757, which controls ion-permeation properties. The N-acetylglucosamine (NAG) groups that were modelled onto residues N769, N1030, N1037 and N2642 are labelled.

Extended Data Fig. 5 The structure of PIEZO2 and the THU organization.

a, A protomer structure of PIEZO2 is presented in a format in which its transmembrane helices roughly adopt a planar configuration. The extracellular loops and intracellular membrane-parallel helices are shown in surface electrostatic potential. Notably, except THU1, which has a short N terminus of seven residues, each of the other eight THUs is preceded by a membrane-parallel helix, which connects perpendicularly to the first transmembrane helices. These intracellular membrane-parallel helices (including TM5pre-α, TM9pre-α, TM13pre-α, TM17pre-α, TM21pre-α, TM25pre-α, TM29pre-α, claspα1–α2 and anchorα3) collectively form an intracellular helical layer with the hydrophobic side facing the membrane side. The enlarged view of the EL7–8, EL11–12, EL15–16, EL19–20 and EL23–24 illustrates the flattened arrangement that is formed by these extracellular loops leaning against each other, except that EL19–20 sits on top of EL23–24. Such an organization might help to stabilize the blade and facilitate conformational propagation from the distal blade to the central region. b, THU5 is used as a typical example to show the organization pattern of the left-handed bundle of four transmembrane helices and the preceding membrane-parallel helix. c, Overlay of THU2 and THU4–THU7 (colour-coded as in a), showing the similar folding pattern. d, Intertwined interaction of the distal beam domain, THU7 and the clasp domain. The residues involved in forming polar interactions are shown in yellow. THU7 is shown in surface representation. The distal part of the beam is apparently kinked at the position of S1466 and buried within a space enclosed by the intracellular side of THU7 and the second and third α-helices of the clasp (claspα2–α3). The clasp domain is composed of two long membrane-parallel helices (claspα1–α2) to form an L-shaped helical structure, and a short helix (claspα3) that is positioned underneath the kink position of the beam. The beam and clasp domains are intertwined together with hydrogen-bond interactions between D1457 and R1702, S1466 and R1717, and R1467 and E1701 for stabilization. The 280 unresolved residues (1728–1947) that link the claspα3 and TM29pre-α1–α2 helices (indicated by the red dashed line) might provide additional interactions and regulation at the distal end of the beam. e, Bottom view of the trimeric central region comprising the beam, the CTD and the latch domain. Two subunits are presented in surface electrostatic potential, and the other subunit is shown in ribbon representation. The proximal end of the beam directly contacts the hairpin-like CTD positioned on top, and connects to the perpendicularly crossed latch domain through 42 unresolved residues (1513–1556). f, Ribbon diagram showing positively charged (blue) and negatively charged (red) residues that contribute to the negative surface potential of the latch domain and positive surface potential of the beam and CTD (as shown in e). The C-terminal section of the latch domain is rich in negatively charged and polar residues (1572-ETDSEE-1577) and is sandwiched in between the beam and the CTD with clusters of positively charged residues (R1500, R1504, K1507 and K1512 in the beam and K2815, R2818 and K2820 in the C-terminal tail). Y1568 in the latch domain points towards the putative intracellular exit of the central pore and forms hydrogen bonds with E2811, which contributes to the cytosolic constriction neck. g, Ribbon diagram showing the intertwined polar interactions of the indicated structural domains. The long membrane-parallel anchorα3 sits right on top of the CTD-hairpin plane and connects to the outer helix through a lysine-rich anchor–outer-helix linker (2456-KRYPQPRGQKKKK-2468), which forms interactions with the polar-residue-rich anchorα2–α3 turn (2426-TDTTT-2430), the glutamate-rich region of the CTDα1–α2-turn (2789-ETGELELEED-2798) and the N-terminal section of the latch domain. Several pairs of hydrogen bonds, including D2427–K2465, T2428–E2796 and T2429–E2797, might help to facilitate the intertwined interactions. The corresponding anchor–outer-helix linker in PIEZO1 is critical for mediating regulation by the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), which binds to both PIEZO1 and PIEZO2.

Extended Data Fig. 6 The central ion-conducting pore and lateral portals of PIEZO2.

a, Ribbon diagram showing the pore module (comprising the outer helix, cap, inner helix and CTD) together with the anchor, latch and beam domains. The central solvent-accessible pathway is marked with a dotted mesh (purple, green and red) generated by the program HOLE (pore radius: red, <1 Å; green, <2 Å; purple, >2 Å). The extracellular vestibule (EV), membrane vestibule and intracellular vestibule are labelled. b, Pore radius along the central axis of the ion-conduction pathway of PIEZO2. The residues that form constriction sites are labelled. c, Representative traces of poking-evoked whole-cell currents in PIEZO1-KO-HEK cells transfected with the indicated constructs. Traces were recorded in an intracellular solution containing 149 mM caesium methanesulfonate and 1 mM CsCl, and an extracellular solution containing 50 mM calcium gluconate and 0.5 mM CaCl2. Currents were elicited from −30 to +30 mV with a change of 10 mV at every step. The red trace represents the current elicited at 0 mV. The number of recorded cells is shown in e. d, Linear regression fit of average I–V relationships of whole-cell currents of the indicated constructs recorded under the conditions described in c. The liquid junction potential of 7.1 mV was subtracted. The number of recorded cells is shown in e. e, Scatter plots of PCa/PCs of the indicated constructs. Data are mean ± s.e.m. and the number of cells tested is labelled. One-way ANOVA: P (PIEZO1 versus PIEZO2) = 0.8323 (not significant; NS); ****P (PIEZO2 versus PIEZO2(E2757A)) < 0.0001. f, Scatter plots of PCa/PCs of the indicated constructs. Data are mean ± s.e.m. and the number of cells tested is labelled. Two-tailed unpaired Student’s t-test: P (PIEZO1 versus PIEZO1(E2537A)) = 0.4353 (NS); P (PIEZO2 versus PIEZO2(E2811A)) = 0.5566 (NS). g, Representative stretch-evoked single-channel current traces recorded at the indicated holding voltages in PIEZO1-KO-HEK cells transfected with constructs expressing either PIEZO2 (seven cells recorded with currents) or PIEZO2(E2757A) (seven cells recorded with currents). h, Histogram analysis of the single-channel conductance from current traces recorded at −140 mV. i, Linear plots of the I–V relationships of the indicated constructs. j, Scatter plots of the slope of the single-channel conductance of the indicated constructs. Data are mean ± s.e.m. and the number of cells tested is labelled. Two-tailed unpaired Student’s t-test: ****P (PIEZO2 versus PIEZO2-E2757A) < 0.0001. k, Surface electrostatic potential representation of the inner helix–CTD-enclosed central pore of PIEZO2 and PIEZO1, showing the hydrophobic transmembrane pore, intracellular fenestration site and the lateral negative surface potential that is contributed by the indicated negatively charged residues (which determine the properties of the pore). Notably, PIEZO1 has apparent membrane-facing cavities (indicated by the green dashed line) between two inner helices. l, m, Surface electrostatic potential representations of the inner helix–CTD-enclosed central pore together with the surrounding anchor, beam and latch domains, showing the intracellular fenestration sites and lateral portals in either a side-view (l) or top-view (m) section at the position indicated by the arrow in panel l. The fenestrations and lateral portals are outlined with yellow dashed lines.

Extended Data Fig. 7 Structural comparison between PIEZO1 and PIEZO2.

a, b, Side (a) or top (b) overlay view of PIEZO2 (salmon), PIEZO1 (PDB: 6B3R) (cyan), PIEZO1 (PDB: 5Z10) (green) and PIEZO1 (PDB: 6BPZ) (purple). c, An enlarged view of the cap and blade regions zoomed in at the capα1–α2 position. Red arrows indicate the shifted positions of PIEZO1 relative to PIEZO2. The clockwise twist and upward displacement of the cap domain is more robust in PIEZO1 (PDB: 6B3R) and PIEZO1 (PDB: 6BZ) than in PIEZO1 (PDB: 5Z10), whereas the lateral displacement of the distal blade of TM13–TM24 is relatively stronger in PIEZO1 (PDB: 5Z10) than in PIEZO1 (PDB: 6B3R). TM13–TM24 of PIEZO1 (PDB: 6PBZ) were not modelled. d, Pore radius along the inner helix–CTD central axis of the ion-permeation pore of the indicated PIEZO2 and PIEZO1 structures. The residues that form constriction sites are labelled. Notably, the transmembrane gates of all three PIEZO1 structures determined in different detergents including GDN (PDB: 6B3R), digitonin (PDB: 6BPZ) and C12E10 (PDB: 5Z10) have dilated transmembrane gates relative to that of PIEZO2 determined in GDN. Furthermore, dilation of the transmembrane gate of PIEZO1 is closely correlated with the displacement of the cap among the three PIEZO1 structures (a–d). For example, compared to PIEZO1 (PDB: 5Z10), PIEZO1 (PDB: 6B3R) and PIEZO1 (PDB: 6BPZ) show relatively stronger displacement of the cap domain (for example, the capα1–α2 region) (c) and concurrent larger expansion of the upper transmembrane gate of L2469 (d). Given that PIEZO2 is much less responsive to stretch stimulation, we speculate that surface tension generated during cryo-EM sample preparation might lead to opening of the transmembrane pore of PIEZO1, but not PIEZO2. e–g, Western blotting of the biotinylated or whole-cell lysate samples derived from HEK293T cells transfected with the indicated constructs using the indicated antibodies. The PIEZO2 antibody in f was raised against the peptide corresponding to residues 2662–2674 in the cap domain of mouse PIEZO2, and could not recognize the PIEZO2(Δcap) protein (data not shown). Thus, the anti-Flag antibody was used to detect the PIEZO2-mRuby2-Flag and PIEZO2(Δcap)-mRuby2-Flag proteins in g. The anti-β-actin antibody detected β-actin for a loading control. The experiment was independently repeated twice or three times with similar results.

Extended Data Fig. 8 Structural comparison between PIEZO1 (PDB: 6B3R) and PIEZO2.

a, c, d, g, Top views of the superimposed PIEZO2 (salmon) and PIEZO1 (PDB: 6B3R) (cyan) structures zoomed in at either capα1–α2 (a), the PIEZO2(L2743) residue (c), the PIEZO2 (F2754) residue (d) or the cytosolic constriction site of PIEZO2(M2767/P2810/E2811) (g). The boxed regions at the centre of a, c and d are presented in enlarged views in Fig. 4a–c, respectively. Arrows indicate the displacement direction of PIEZO1 relative to PIEZO2; red arrows show the displacement of the cap or the blade domains, whereas purple arrows show the displacement of the side chain of the indicated residues. For example, TM16 of PIEZO1 has a lateral displacement of about 12 Å relative to that of PIEZO2 (a). b, e, Side views of the superimposed PIEZO2 (salmon) and PIEZO1 (PDB: 6B3R) (cyan) trimers (b) or monomers (e). Red arrows indicate the displacement direction of PIEZO1 relative to PIEZO2. The blue and red dashed lines in e indicate the flatness of the curved transmembrane region of the blade. f, The upward displacement associated with the beam, CTD, anchor and outer helix might contribute to the upward displacement of the cytosolic constriction neck, the transmembrane gate and the inner helix.

Extended Data Fig. 9 Mapping disease-causing mutations in human PIEZO1 and PIEZO2 onto the mouse PIEZO2 structure.

The dots in different colours represent the disease-causing residues labelled in the protein sequence shown in Supplementary Fig. 2.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
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Wang, L., Zhou, H., Zhang, M. et al. Structure and mechanogating of the mammalian tactile channel PIEZO2. Nature 573, 225–229 (2019). https://doi.org/10.1038/s41586-019-1505-8

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