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Architecture of the mammalian mechanosensitive Piezo1 channel

Nature volume 527, pages 64–69 (2015)Cite this article

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Abstract

Piezo proteins are evolutionarily conserved and functionally diverse mechanosensitive cation channels. However, the overall structural architecture and gating mechanisms of Piezo channels have remained unknown. Here we determine the cryo-electron microscopy structure of the full-length (2,547 amino acids) mouse Piezo1 (Piezo1) at a resolution of 4.8 Å. Piezo1 forms a trimeric propeller-like structure (about 900 kilodalton), with the extracellular domains resembling three distal blades and a central cap. The transmembrane region has 14 apparently resolved segments per subunit. These segments form three peripheral wings and a central pore module that encloses a potential ion-conducting pore. The rather flexible extracellular blade domains are connected to the central intracellular domain by three long beam-like structures. This trimeric architecture suggests that Piezo1 may use its peripheral regions as force sensors to gate the central ion-conducting pore.

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Figure 1: Piezo1 forms a homotrimer.
Figure 2: Overall structure of Piezo1.
Figure 3: Organization of the transmembrane skeleton.
Figure 4: Putative ion-conducting pore.
Figure 5: Conformational heterogeneity of the ‘blade’ and a proposed model of force-induced gating of Piezo channels.

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Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The 3D cryo-electron microscopy density map has been deposited in the Electron Microscopy Data Bank (EMDB), with accession code EMD-6343. The coordinates of atomic models have been deposited in the Protein Data Bank (PDB) under the accession codes 4RAX for the CED and 3JAC for the full length.

Change history

  • 04 November 2015

    A grant number in the Acknowledgements was corrected.

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Acknowledgements

We thank H. Yu and J. Chai for discussion and proofreading of the manuscript. We thank the staff at beamline BL17U of the Shanghai Synchrotron Radiation Facility (SSRF) and beamline 3W1A of the Beijing Synchrotron Radiation Facility (BSRF) for their assistance in data collection. K. Wu and H. Wang are acknowledged for technique help. We also thank the National Center for Protein Sciences (Beijing, China) for technical support with cryo-electron microscopy data collection and for computation resources. This work was supported by grants from the Ministry of Science and Technology (2012CB911101 and 2011CB910502 to M.Y., 2015CB910102 to B.X. and 2013CB910404 to N.G.), the National Natural Science Foundation of China (21532004, 31570733, 31030020 and 31170679 to M.Y., 31422016 to N.G. and 31422027 to B.X.) and the Ministry of Education (the Young Thousand Talent program to B.X.).

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Author notes

  1. Jingpeng Ge, Wanqiu Li, Qiancheng Zhao and Ningning Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences or Medicine, Tsinghua University, Beijing, 100084, China

    Jingpeng Ge, Qiancheng Zhao, Maofei Chen, Ruochong Li, Bailong Xiao & Maojun Yang

  2. Ministry of Education, Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China

    Jingpeng Ge, Wanqiu Li, Ningning Li, Maofei Chen, Ruochong Li, Ning Gao & Maojun Yang

  3. Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing, 100084, China

    Qiancheng Zhao, Peng Zhi & Bailong Xiao

  4. IDG/McGovern Institute for Brain Research, Tsinghua University, Beijing, 100084, China

    Bailong Xiao

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  1. Jingpeng Ge
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Contributions

M.Y. directed the study. J.G., M.C. and R.L. performed protein purification, detergent screening and crystallization. W.L. performed electron microscopy sample preparation, data collection and structural determination with N.L; Q.Z. was responsible for molecular cloning (with P.Z.), protein purification, detergent screening and biochemical and confocal imaging studies. N.G. directed electron microscopy studies and wrote part of the manuscript. B.X. initiated the project and directed molecular cloning, protein expression and purification and wrote most of the manuscript. All authors contributed to discussion of the data and editing of the manuscript.

Corresponding authors

Correspondence to Ning Gao, Bailong Xiao or Maojun Yang.

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Extended data figures and tables

Extended Data Figure 1 Biochemical characterization of the recombinant protein of Piezo1–pp–GST.

a, A representative trace of gel filtration chromatography of the Piezo1–pp–GST protein. b, Protein samples of the indicated fractions were subjected to SDS–PAGE and Coomassie blue staining. Fractions of 8.0 ml and 8.5 ml (elution volume) were used for the negative-staining electron microscopy and native gel analyses, respectively.

Extended Data Figure 2 Negative-staining electron microscopy examination of Piezo1 in different detergents.

a, A representative micrograph of negatively stained Piezo1 purified with C12E10. b, 2D class averages of Piezo1 particles (C12E10). c, A representative micrograph of negatively stained Piezo1 purified with C12E8. d, 2D class averages of Piezo1 particles (C12E8). e, A representative micrograph of negatively stained Piezo1, with amphipol A8-35 as detergent. f, 2D class averages of Piezo1 particles (amphipol A8-35).

Extended Data Figure 3 Initial model of Piezo1 generated from the random conical tilt method and validation of the model using cryo-electron microscopy data from a TF20 microscope.

a, b, Representative micrographs of negatively stained Piezo1 in C12E10 collected in random conical tilt (RCT) pairs (a, untilted and b, 50° tilted). c, Top view of an RCT reconstruction, showing an overall threefold symmetry for the Piezo1 complex, is shown on the left. The right-hand side shows the top view of the refined model, obtained by a structural refinement of all particles from untilted micrographs. d, e, Model validation was performed by refinement of cryo-electron microscopy particles collected with TF20, with a Gaussian ball (d) or the RCT model (e) as initial reference. The 3D volumes are shown in top, side and bottom views. During the refinement, both the symmetry-free (C1) and symmetry-imposed (C3) reconstructions were tested. Note that some of these reconstructions have incorrect handedness.

Extended Data Figure 4 Representative raw particles of Piezo1 collected with the Titan Krios electron microscope fitted with a K2 electron detector.

A collection of raw particles of Piezo1 (eluted with C12E10), collected with Titan Krios (300 kV).

Extended Data Figure 5 Workflow of 3D classification of Piezo1 particles.

a, The schematic diagram of a series of 3D classification procedures with RELION is shown (also see Methods). After several rounds of 2D classification, the remaining 120,000 particles were subjected to three rounds of 3D classification without imposing any symmetry. A final set of particles (class 4 after the second round of 3D classification), with its reconstruction best matching threefold symmetry, was subjected to 3D refinement (C3 imposed). Notably, further 3D classification of this class resulted in generally similar structures (vertically arranged panels) without detectable improvement of conformational homogeneity. A top view of the soft mask used in structural refinement is also shown (yellow). b, Distribution of particle orientations in the last iteration of the refinement. c, Gold-standard Fourier shell correlation (FSC) curves of the final density map. The FSC curves were calculated with (red) or without (blue) the application of a soft mask to the two half-set maps. The final FSC curve (red) was corrected for the soft-mask-induced effect. Reported resolutions were based on FSC = 0.143 criteria.

Extended Data Figure 6 The trimeric CEDs form the cap domain of Piezo1.

a, A representative micrograph of negatively stained Piezo1(ΔCED) in C12E10. b, 2D class averages of negatively stained Piezo1(ΔCED) particles. It is evident that the central cap domain is absent from these average images. c, Sequence alignment of the CED region of Piezo1 from Mus musculus and Caenorhabditis elegans. Identical residues are highlighted in blue. Secondary structures are indicated by cartoons above the primary sequence. Sequence alignment was performed using Clustal W2 (ref. 71). d, Structure alignment of the trimeric CED of Piezo1 from M. musculus and C. elegans. The three CEDs are coloured in purple, cyan and green, respectively. The CED of C. elegans is coloured in orange. e, A representative trace of gel filtration of the CED of Piezo1. The molecular weights are labelled. Protein samples of the indicated fractions were subjected to SDS–PAGE and Coomassie blue staining (bottom). f, Transparent surface representation of the segmented density map of the cap, superimposed with the trimeric CED crystal structure. The trimeric CEDs are coloured as in d.

Extended Data Figure 7 Local resolution map of the final density map.

a, The final 3D density map of Piezo1 is coloured according to the local resolutions estimated by the software of ResMap. The density map is shown in three different views (top, bottom and side, respectively). b, The final 3D density map (transparent) is superimposed with a poly-alanine model and the crystal structure of the trimeric CED. Three protomers are coloured cyan, purple and green, respectively.

Extended Data Figure 8 Density connections between the transmembrane helices and between the helices in the compact CTD.

a, Alanine models of five representative pairs of transmembrane helices are displayed with their densities (mesh) superimposed. The transmembrane region is highlighted by a light purple shade with the intracellular and extracellular sides indicated. b, An alanine model of the anchor motif with its density superimposed (mesh). Four helices (α1anchor–α4anchor) connecting PH1 and OH are labelled. The transmembrane region is highlighted by a light purple shade with the intracellular and extracellular sides indicated. c, An alanine model of the last four helices (α1CTD–α4CTD) of the trimeric CTD, superimposed with the density of the CTD (mesh).

Extended Data Figure 9 Secondary structure analyses of the C-terminal segments of Piezo1 proteins from different species.

Sequence alignment of the C-terminal regions of Piezo1 from different species. The alignment was performed using Clustal W2 (ref. 71). The anchor motif and the CTD are highlighted in green and pink, respectively. For clarification, the sequences of CEDs were omitted and are indicated by red dashed lines. Secondary structures (α-helices) predicted with PredictProtein72 are shown as black lines. Transmembrane segments were predicted using multiple web servers including Topcons73 (green lines), TMHMM2 (ref. 74) (blue lines), HMMTOP75 (orange lines) and Phobius76 (pink lines).

Extended Data Table 1 Statistics of data collection and structure refinement.
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Ge, J., Li, W., Zhao, Q. et al. Architecture of the mammalian mechanosensitive Piezo1 channel. Nature 527, 64–69 (2015). https://doi.org/10.1038/nature15247

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