Abstract
TRPML3 channels are mainly localized to endolysosomes and play a critical role in the endocytic pathway. Their dysfunction causes deafness and pigmentation defects in mice. TRPML3 activity is inhibited by low endolysosomal pH. Here we present cryo-electron microscopy (cryo-EM) structures of human TRPML3 in the closed, agonist-activated, and low-pH-inhibited states, with resolutions of 4.06, 3.62, and 4.65 Å, respectively. The agonist ML-SA1 lodges between S5 and S6 and opens an S6 gate. A polycystin-mucolipin domain (PMD) forms a luminal cap. S1 extends into this cap, forming a 'gating rod' that connects directly to a luminal pore loop, which undergoes dramatic conformational changes in response to low pH. S2 extends intracellularly and interacts with several intracellular regions to form a 'gating knob'. These unique structural features, combined with the results of electrophysiological studies, indicate a new mechanism by which luminal pH and other physiological modulators such as PIP2 regulate TRPML3 by changing S1 and S2 conformations.
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Acknowledgements
This work was supported by the National Basic Research Program of China (grant 2014CB910301 to J.Y.), the National Institutes of Health (grant R01GM085234 to J.Y.), the National Natural Science Foundation of China (grant 31370821 to J.Y.; grant 31570730 to X.L.), the National Key Research and Development Program (grants 2016YFA0501102 and 2016YFA0501902 to X.L.), the Top Talents Program of Yunnan Province (grant 2011HA012 to J.Y.), the High-level Overseas Talents of Yunnan Province (J.Y.), the China Youth 1000-Talent Program of the State Council of China (X.L.), Beijing Advanced Innovation Center for Structural Biology (X.L.), and the Tsinghua-Peking Joint Center for Life Sciences (X.L.).
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M.L. and J.Y. conceived and initiated the project. X.Z., M.L., D.S., Q.J., H.L., X.L., and J.Y. designed the experiments, analyzed the results, and wrote the manuscript. M.L. performed all molecular biology and biochemical experiments and built the atomic models. X.Z. and X.L. performed all cryo-EM experiments, including data acquisition and processing. D.S., Q.J., and H.L. performed electrophysiology experiments. All authors contributed to manuscript discussion, preparation, and editing.
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Supplementary Figure 1 Amino acid sequence alignment of human TRPML subunits.
Secondary structural elements are marked according to the pH 7.4 ML-SA1-bound TRPML3 structure. Green and yellow highlight identical and similar amino acids, respectively. The ion selectivity filter is boxed in red. The luminal pore-loop is boxed in blue, with the luminal pore-loop aspartates marked by red triangles. Red and green circles mark amino acids involved in binding PI(4,5)P2 and PI(3,5)P2, respectively. H283 is indicated by a red star. Orange circles mark amino acids involved in binding ML-SA1. Yellow star marks A419.
Supplementary Figure 2 Single-particle cryo-EM analysis of apo-TRPML3 at pH 7.4.
(a) A representative motion-corrected micrograph. Typical particles are marked with orange boxes. (b) Fourier power spectrum of the micrograph shown in a. (c) Gallery of typical two-dimensional class averages. (d) Flow chart of cryo-EM data processing. (e) Euler angle distribution of all particles used in the final map reconstruction. Each orientation is represented by a cylinder, for which both the height and color (from blue to red) are proportional to the number of particles for that specific direction. (f) Local resolution of the cryo-density map. (g) The gold-standard FSC curve of the final reconstruction (black) and the FSC curve between the final reconstruction and the map calculated from the atom model (blue). (h) Model validation. Blue, model versus the summed map. Black, model versus half 1 map (called ‘work’, used for model refinement). Red, model versus half 2 map (called ‘free’, not used for model refinement).
Supplementary Figure 3 Single-particle cryo-EM analysis of ML-SA1-bound TRPML3 at pH 7.4.
(a) A representative motion-corrected micrograph. Typical particles are marked with orange boxes. (b) Fourier power spectrum of the micrograph shown in a. (c) Gallery of typical two-dimensional class averages. (d) Flow chart of cryo-EM data processing. (e) Euler angle distribution of all particles used in the final map reconstruction. Each orientation is represented by a cylinder, for which both the height and color (from blue to red) are proportional to the number of particles for that specific direction. (f) Local resolution of the cryo-density map. (g) The FSC curve of the final reconstruction (black) and the FSC curve between the final reconstruction and the map calculated from the atom model (blue). (h) Model validation. Blue, model versus the summed map. Black, model versus half 1 map (called ‘work’, used for model refinement). Red, model versus half 2 map (called ‘free’, not used for model refinement).
Supplementary Figure 4 Single-particle cryo-EM analysis of apo-TRPML3 at pH 4.8.
(a) A representative motion-corrected micrograph. Typical particles are marked with orange boxes. (b) Fourier power spectrum of the micrograph shown in a. (c) Gallery of typical two-dimensional class averages. (d) Flow chart of cryo-EM data processing. (e) Euler angle distribution of all particles used in the final map reconstruction. Each orientation is represented by a cylinder, for which both the height and color (from blue to red) are proportional to the number of particles for that specific direction. (f) Local resolution of the cryo-density map. (g) The FSC curve of the final reconstruction (black) and the FSC curve between the final reconstruction and the map calculated from the atom model (blue). (h) Model validation. Blue, model versus the summed map. Black, model versus half 1 map (called ‘work’, used for model refinement). Red, model versus half 2 map (called ‘free’, not used for model refinement).
Supplementary Figure 5 Representative cryo-EM density maps.
(a) Cryo-EM density maps and atomic models of selected regions of TRPML3 in the ML-SA1-bound pH 7.4 condition (left) or the apo pH 4.8 condition (right). The ML-SA1-bound pH 7.4 maps were low-pass filtered to 3.62 Å and amplified by a temperature factor of -180 Å2, and were contoured at 3.0σ. The apo pH 4.8 maps were low-pass filtered to 4.65 Å and amplified by a temperature factor of -244 Å2, and were contoured at 4.0σ. (b) Comparison of the cryo-EM density map in a crevice surrounded by S5, S6 and pore helix 1 in the apo pH 7.4 (left, filtered to 4.06 Å and contoured at 3.0σ) and ML-SA1-bound pH 7.4 (middle, filtered to 3.62 Å and contoured at 3.0σ) structures. The right panel shows the normalized different density map between the two structures (filtered to 6 Å and contoured at 15.0σ).
Supplementary Figure 6 Structure of the TRPML3 PMD compared with that of the TRPML1 PMD.
(a) Electrostatic-potential surface representation of the TRPML3 PMD, viewed from the luminal side of the membrane (left) or parallel to the membrane (right). (b) Superposition of TRPML3 and TRPML1 PMDs. (c). Superposition of the backbone α carbons of the two luminal pore-loops in stereo view. Same color representation as in b. The first amino acid of each luminal pore-loop is numbered 1. (d) Stereo view of the TRPML3 luminal pore-loop. (e) Stereo view of the TRPML1 luminal pore-loop.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6 (PDF 2571 kb)
Life Sciences Reporting Summary
Original electrophysiological data for Figures 2 and 5 (PDF 130 kb)
Supplementary Data Set 1
Source data for Figures 2 and 5. (XLSX 95 kb)
Conformational changes induced by the binding of ML-SA1
ML-SA1 binding causes many movements. For example, when viewed from the side, S5 and S6 move outward and the S4-S5 linker moves downward by 2 to 4 Å, the pore-loop moves downward by ∼2 Å, and S6 undergoes a 27 degree counterclockwise rotation. The zoom-in view from the bottom shows the movement of I498 (in space-filling form) between the closed state and open state upon ML-SA1 binding and unbinding. (MOV 23233 kb)
Conformational changes induced by pH changes
The movie shows a morph between the pH 7.4 apo structure and the pH 4.8 apo structure. The channel is viewed first from the side (parallel to the membrane) and then from top down (perpendicular to the membrane). (MOV 16106 kb)
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Zhou, X., Li, M., Su, D. et al. Cryo-EM structures of the human endolysosomal TRPML3 channel in three distinct states. Nat Struct Mol Biol 24, 1146–1154 (2017). https://doi.org/10.1038/nsmb.3502
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DOI: https://doi.org/10.1038/nsmb.3502
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