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Research Article
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Structure of human Dispatched-1 provides insights into Hedgehog ligand biogenesis

View ORCID ProfileHongwen Chen, View ORCID ProfileYang Liu, View ORCID ProfileXiaochun Li  Correspondence email
Hongwen Chen
1Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA
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Yang Liu
1Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA
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Xiaochun Li
1Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA
2Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA
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  • For correspondence: xiaochun.li@utsouthwestern.edu
Published 9 July 2020. DOI: 10.26508/lsa.202000776
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  • Figure 1.
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    Figure 1. Functional validation of DISP1* for structural investigation.

    (A) A diagram of topology of human DISP1. Residues 1–171 and 1,249–1,524 were removed in DISP1*. (B) Experimental scheme to measure SHH release capacity. HEK293 Flp-In T-REX cells were stably transfected with an inducible 3× Flag-tagged Scube2. Cells were induced and the supernatant containing Scube2 conditioned medium was collected, filtered, and mixed with fresh medium at a 1:1 ratio and finally transferred to DISP1−/− or DISP1* and DISPFL rescued MEF cells stably expressing FL-SHH. After 48 h of incubation, the SHH-N conditioned medium was harvested and mixed with fresh medium at 1:1 ratio to incubate with SHH-Light II cells. (C) SHH-N release in DISP1−/− MEF cells that were transduced with both SHH and empty vector (DISP1KO), full-length DISP1 (DISP1FL) or DISP1* was checked via dual luciferase assay where the SHH-Light II cells stably express firefly luciferase with an 8×-Gli promoter and Renilla luciferase with a constitutive promoter. Data are mean ± SD (n = 8 biologically independent experiments). (D) Pull-down assay of native SHH-N with DISP1*-WT or DISP1*–NNN mutant detected by Coomassie staining.

  • Figure S1.
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    Figure S1. Expression and purification of human DISP1* protein with SHH-N ligand.

    (A) Representative Superose 6 increase 10/30 gel-filtration chromatogram of DISP1*. Peak fraction of DISP1* is shown on SDS–PAGE with molecular markers. (B) Representative Superose 6 increase 10/30 gel-filtration chromatogram of DISP1*–SHH-N complex. Peak fraction of the complex is shown on SDS–PAGE with molecular markers.

  • Figure S2.
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    Figure S2. Verification of DISP1 transduction by RT-qPCR.

    DISP1−/− MEF cells that stably expressed SHH were transduced with empty vector (DISP1KO), full-length DISP1 (DISP1FL) or DISP1*. Overexpression of DISP1FL and DISP1* was verified by RT-qPCR. Data are mean ± SD (n = 5 technically independent samples).

  • Figure S3.
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    Figure S3. Data processing of DISP1*.

    (A) Data processing workflow. A representative electron micrograph at approximately −1.5-μm defocus is shown with a scale bar of 20 nm. The cryo-EM 3D classification from RELION and the masks used for the refinement are shown. The final cryo-EM map after RELION-3 refinement was sharpened using postprocess with a B-factor value of −180 Å2. (B) Fourier shell correlation curve as a function of resolution using RELION-3 output.

  • Figure S4.
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    Figure S4. Data processing of DISP1*–HH-N.

    (A) Data processing workflow. The cryo-EM 3D classification from RELION and the masks used for the refinement are shown. The putative SHH-N is indicated by dashed circle. (B) Fourier shell correlation curve as a function of resolution using RELION-3 output.

  • Figure 2.
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    Figure 2. Overall Structure of DISP1*.

    (A) Cryo-EM map after final RELION-3 refinement sharpened using “post-processing.” (B) Overall structure showing DISP1* viewed from the side of the membrane. (C) Ribbon representations of the structure viewed from extracellular and cytosolic side, respectively. Residues that are closed to the Furin cleavage site, as well as transmembrane helices with the putative cholesteryl hemisuccinate (yellow sticks) are labeled.

  • Figure S5.
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    Figure S5. Cryo-EM map of structural elements in DISP1*.

    (A) The Fourier shell correlation curves calculated between the refined structure and the half map used for refinement (green), the other half map (orange), and the full map (blue). (B) Cryo-maps of structure colored by local resolution estimated using ResMap. (C) The major helices of DISP1* at 5σ level. EM map and model of the complex are shown in mesh and cartoon.

  • Figure 3.
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    Figure 3. Structural comparison of DISP1* with PTCH1 and NPC1.

    (A) Structural comparison of DISP1* with PTCH1 (pdb: 6OEU) and NPC1 (pdb: 5U74). (B) Superposition of extracellular domains (ECDs) of DISP1* with the counterparts of PTCH1 and NPC1, respectively. A 15-Å shift of ECD-I of DISP1* from ECD-II is labeled. (C) cryo-EM map of DISP1* complex with SHH-N from Relion-3 output. (D) Ribbon representation of the complex structure. The putative SHH-N ligand is shown as a surface model in blue. (E) The structure of PTCH1 with its ligand SHH-N (pdb: 6E1H).

  • Figure S6.
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    Figure S6. Sterol-like densities in the SHH-N–mediated PTCH1 dimer.

    Sterol-like maps at 5σ level at 3.5 Å resolution in the domains of extracellular domains, the sterol-sensing domains, and near TM-12 are colored in green, red and purple, respectively.

  • Figure 4.
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    Figure 4. The transmembrane domain of DISP1* with sterol-like ligands.

    (A) Structural comparison of the transmembrane domains of DISP1* with PTCH1. (B) The four sterol-bound sites in the TMs of DISP1*. The location of the three Asp residues is indicated by a red dashed circle. EM maps and models of cholesteryl hemisuccinate are shown at 5σ level in mesh and cartoon. (C) The putative sterol in site 4 triggers the conformational changes of TMs. Electrostatic surface representation is shown in the right panel. (D, E) The structural comparison shows that the sterol in site 4 triggers a conformational change of TM10 to provide the cytoplasmic mobility between the N-half TMs and C-half TMs.

  • Figure S7.
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    Figure S7. Density map of Drosophila DISP in complex with its unmodified ligand HH-N.

    Cryo-EM map of Drosophila DISP–HH-N complex at 2σ level at 4.8 Å resolution (EMD-10464). The model of the complex (pdb: 6TD6) is fitted into the density map and HH-N is colored in blue.

Supplementary Materials

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  • Table S1 Cryo-EM data collection, refinement, and validation statistics.

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Structure of human dispatched-1
Hongwen Chen, Yang Liu, Xiaochun Li
Life Science Alliance Jul 2020, 3 (8) e202000776; DOI: 10.26508/lsa.202000776

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Structure of human dispatched-1
Hongwen Chen, Yang Liu, Xiaochun Li
Life Science Alliance Jul 2020, 3 (8) e202000776; DOI: 10.26508/lsa.202000776
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Volume 3, No. 8
August 2020
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