Abstract
The activities of organellar ion channels are often regulated by Ca2+ and H+, which are present in high concentrations in many organelles. Here we report a structural element critical for dual Ca2+/pH regulation of TRPML1, a Ca2+-release channel crucial for endolysosomal function. TRPML1 mutations cause mucolipidosis type IV (MLIV), a severe lysosomal storage disorder characterized by neurodegeneration, mental retardation and blindness. We obtained crystal structures of the 213-residue luminal domain of human TRPML1 containing three missense MLIV-causing mutations. This domain forms a tetramer with a highly electronegative central pore formed by a novel luminal pore loop. Cysteine cross-linking and cryo-EM analyses confirmed that this architecture occurs in the full-length channel. Structure–function studies demonstrated that Ca2+ and H+ interact with the luminal pore and exert physiologically important regulation. The MLIV-causing mutations disrupt the luminal-domain structure and cause TRPML1 mislocalization. Our study reveals the structural underpinnings of TRPML1's regulation, assembly and pathogenesis.
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Acknowledgements
We thank Y. Chen and M. Chalfie (Columbia University) for providing the C. elegans cDNA library; R. Prywes (Columbia University) for providing HeLa cells; T. Patel and S. Banta for help with the CD experiment; and the staff at X29 of the National Synchrotron Light Source, Brookhaven National Laboratory, for synchrotron support. This work was supported by grants to J.Y. from the National Key Basic Research Program of China (2014CB910301), the National Institutes of Health (R01GM085234 and RO1NS053494), the National Natural Science Foundation of China (31370821), the Top Talents Program of Yunnan Province (2011HA012) and the High-level Overseas Talents of Yunnan Province; and grants to X.L. from the China Youth 1000-Talent Program of the State Council of China, the Beijing Advanced Innovation Center for Structural Biology, the Tsinghua-Peking Joint Center for Life Sciences and the National Natural Science Foundation of China (31570730).
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M.L. and J.Y. conceived and initiated the project. M.L. obtained the first crystal structure at pH 6.0 and contributed to most of the other experiments. W.K.Z. performed most of the electrophysiology experiments. N.M.B. obtained the crystal structures at pH 4.5 and 7.5 and performed the imaging experiments. L.T. helped supervise X-ray crystallography data collection and atomic-model building. X.Z. and X.L. performed the cryo-EM experiments and data processing. D.S., H.L., S.W. and I.E.M. performed experiments or analysis. All authors contributed to manuscript preparation and editing. M.L., W.K.Z., N.M.B. and J.Y. wrote the paper.
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Integrated supplementary information
Supplementary Figure 1 Amino acid sequence alignment of TRPML subunits.
(a) Subcellular localization and transmembrane topology of TRPML1. (b) Amino acid sequence alignment of TRPML subunits. Green and yellow mark identical and similar amino acids, respectively. Putative S1-S6 segments are indicated in gray. Secondary structures of TRPML1 I-II linker are colored in the same scheme as in Fig. 2c. Bold black residues boxed in red are involved in intersubunit interactions. Red triangles mark the luminal pore-loop aspartates. Stars mark the amino acids whose point mutation causes MLIV.
Supplementary Figure 2 A docking model of the TRPML1 I–II linker.
The TRPML1 I-II linker structure is visually and manually docked onto the transmembrane domain (TMD) of the TRPV1 structure (PDB code: 3J5P). (a,c) and (b,d) show the ribbon and surface representations, respectively, of the docked structures. Upper panels, top down views from the extracellular/luminal side of the membrane. Lower panels, side views parallel to the membrane. The α1 helix of the TRPML1 I-II linker and S1 of TRPV1 are highlighted in red and gold, respectively, in (a,c).
Supplementary Figure 3 Single-particle cryo-EM analysis of TRPML1.
(a) A representative micrograph. Typical particles are marked with yellow boxes. (b) Fourier power spectrum of the micrograph shown in a with the Thon ring extending to 3Ǻ. (c) Outline of image processing. After multiple rounds of 2D and 3D classification to remove unwanted particles, 71,052 particles were selected from 201,010 autopicked particles and subjected to refinement with C4 symmetry imposed, yielding a reconstruction at a resolution of 8.12 Å. Due to the possible flexibility of the transmembrane domain (TMD), a soft mask surrounding the more rigid extracellular domain (shown in green) was applied for further refinement, resulting in a reconstruction at a resolution of 5.28 Å. The angular distribution of the final structure is also shown. (d) Enlarged views of representative 2D classes. Orange and green arrows point to TMD regions with different densities, a phenomenon suggestive of flexibility in the TMD. (e) FSC curves of the final TRPML1 reconstruction with (blue) or without (black) mask. The pixel size used was 2.64 Å; thus, the blue curve crossing the Nyquist frequency yields a 5.28 Å resolution.
Supplementary Figure 4 MLIV-causing mutations cause TRPML1 mislocalization.
(a) Location of three MLIV-causing missense mutations in the I-II linker structure, marked in red. (b) Confocal images of live HeLa cells expressing the indicated GFP-tagged channels. Red indicates LysoTracker-labeled lysosomes.
Supplementary Figure 5 Cysteine modification in the luminal pore reduces ion conduction.
(a,b) Time course of whole-cell currents of the indicated channels in response to 5 mM extracellular MTSET. NDF: nominal divalent cation free. (c) Current-voltage curves taken at the time points indicated in b. (d) Normalized and averaged current amplitude of TRPML1vp-3C at -80 mV of at pH 7.4. Number of recordings is indicated inside the bar. Error bars represent SEM. * p<0.05 with Student’s t-test.
Supplementary Figure 6 The luminal-pore aspartate mutations do not affect inward rectification.
(a) Families of TRPML1vp-3DQ currents at the indicated pH 7.4 and extracellular Ca2+ concentrations. (b) Current-voltage relationship of the currents in a.
Supplementary Figure 7 Comparison of the structures of the I–II linkers of TRPML1 and TRPP2.
(a) Amino acid sequence alignment of the TRPML1 and TRPP2 I-II linkers. Secondary structures are indicated. Bold black residues are identical residues in both sequences. Bold red residues are the luminal pore-loop aspartates. (b) Superposition of the I-II linker protomer structures of TRPML1 (pH 6.0) and TRPP2 (PDB code: 5T4D), aligned by the β strands. (c, d) Superposition of the TRPML1 and TRPP2 I-II linker tetramer structures, aligned by the β strands and viewed from above (c) or parallel (d) to the membrane. e, Same view as in (d) but showing only two diagonally opposed subunits, highlighting the different orientations of the luminal pore-loop of TRPML1 and its counterpart in TRPP2.
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Supplementary Data Set 1
Uncropped western blots for Figures 3b, 4b and 5c (PDF 137 kb)
Supplementary Data Set 2
Original data for line figures in Figures 1, 4, 5 and 6 and in Supplementary Figures 5 and 6 (XLSX 40 kb)
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Li, M., Zhang, W., Benvin, N. et al. Structural basis of dual Ca2+/pH regulation of the endolysosomal TRPML1 channel. Nat Struct Mol Biol 24, 205–213 (2017). https://doi.org/10.1038/nsmb.3362
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DOI: https://doi.org/10.1038/nsmb.3362
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