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Structural basis of sterol recognition by human hedgehog receptor PTCH1

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Science Advances  18 Sep 2019:
Vol. 5, no. 9, eaaw6490
DOI: 10.1126/sciadv.aaw6490
  • Fig. 1 Structure of the PTCH1Δ-ShhNC24II complex.

    (A) A schematic representation of the constructs used in this study. For comparison, the wild-type full-length proteins (PTCH1 and ShhN) are shown. (B) The hedgehog pathway activation assay confirms the functionality of the ShhNC24II preparation. NIH 3T3 cells were treated with the indicated concentrations of the ligands, and hedgehog pathway activity was evaluated analyzing Gli1 mRNA levels by qPCR; error bars indicate SD (n = 3). (C) Density map of the reconstituted PTCH1Δ-ShhNC24II complex. (D) The views of the modeled complex corresponding to the views shown in (C). The key components of the complex are labeled, including the TM1–6 (“M1”), TM7–12 (“M2”), and the extracellular domains (ECD1–2). (E and F) Comparison of the structure to the previously solved PTCH1-ShhN (E) and PTCH1:rhShhN 2:1 complex (F). The bound ShhNC24II perfectly aligns to the previously observed complexes mediated by the metal-binding site interface of the ligand.

  • Fig. 2 Bound sterol molecules revealed by the PTCH1Δ-ShhNC24II structure.

    (A) The 3D reconstruction features several well-resolved density elements in the ECD region and surrounding the TM domains of PTCH1Δ (isolated density are colored yellow). The SSD (TM2–6) and the SSDL (TM8–12) are colored orange and blue. Inset: Previously observed sterol sites are indicated as sites 1, 2, and 3; additional partial density is present in the neck region of PTCH1Δ (site 0, pink), suggesting partial occupancy of the site. (B) Focused density map of the TM region (TM map) indicates the well-resolved sites 1, 2, and 4, as well as a number of novel sites, indicated with a mesh; the pocket region within the SSD and the corresponding SSDL is indicated by a rounded rectangle. (C) The CHS molecules were modeled into the well-defined density map regions (B) using 10σ cutoff of the unsharpened refined density map. The closed circles correspond to the novel sterol sites revealed by the PTCH1Δ-ShhNC24II reconstruction. (D and E) Views of the sterols surrounding the SSD and SSDL domains. The protein model is shown as a surface, colored according to atom type; carbon atoms in the SSD and SSDL are colored orange and blue, respectively.

  • Fig. 3 The inner leaflet SSD sterol site points at a functional role in PTCH1.

    (A) The site 5 sterol (as defined in Fig. 2B; colored yellow) is within contact of the residue D599, previously shown to be functionally critical in the SSDs of SCAP and Drosophila Patched. (B) A similar site, unoccupied by a sterol molecule, has been previously observed in the structure of the cholesterol transporter NPC1, homologous to PTCH1. The PTCH1Δ-ShhNC24II structure was aligned to the NPC1 X-ray structure (cyan). Inset: Alignment of the two structures using the residues of TM6 shows a great degree of similarity in the region surrounding the site 5 sterol. (C) Sequence alignment of key SSD-containing proteins, PTCH1, DISP1, NPC1, HMG-CoA reductase, and SCAP, shows the similarity between the elements involved in inner leaflet sterol binding site. The pink arrow indicates the residue D599 [shown in (A)]. The cyan bar corresponds to residues shown with side chains in (B). TM6 and IL3 of PTCH1 are indicated below the sequence alignment.

  • Fig. 4 Sterol binding site overlap observed at the SSD and SSDL domains of PTCH1.

    (A and B) The M1 and M2 regions of PTCH1Δ were aligned using the SSD (orange) and SSDL domains (blue). The SSD and SSDL regions are represented as cartoons; the TM helices TM1 and TM7 are shown as ribbons. Sterols bound at the SSD (yellow) and SSDL regions (light blue) are labeled using corresponding colored circles. The outer leaflet sites 1, 6, and 7 at the SSD and site 10 at the SSDL overlap. The dotted lines in (A) and (B) indicate access of sterols via the inner leaflet to site 5 (SSD) and via the outer leaflet to pocket sites 1, 6, and 7 (SSD) and site 10 (SSDL). (C) Putative model of sterol translocation via PTCH1. Left: In the absence of hedgehog ligand (red), the protein (illustrated using PDB ID: 6D4H) moves the inner leaflet sterols along the path formed by the SSD. Right: Binding of the ligand negatively regulates this activity; the sterols accumulate at the SSD pocket region, evidenced by our 3D reconstruction. Presence of sterols at the SSDL, along with its similarity with SSD, may suggest a similar functional role (marked by the dashed lines).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaaw6490/DC1

    Fig. S1. Purification of the hedgehog ligand and PTCH1.

    Fig. S2. Cryo-EM and single-particle analysis workflow.

    Fig. S3. FSC plots and 3D reconstruction details.

    Fig. S4. Features of the map and the atomic model of the protein.

    Fig. S5. Disease-linked mutations in PTCH1 are present within the SSD and SSDL regions of the protein.

    Fig. S6. Mutations designed to disrupt sterol binding sites in PTCH1 fail to suppress hedgehog pathway activation.

    Movie S1. The density elements corresponding to bound sterols in the PTCH1 TM-focused map.

    Table S1. Cryo-EM data collection, single-particle analysis, and model building statistics.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Purification of the hedgehog ligand and PTCH1.
    • Fig. S2. Cryo-EM and single-particle analysis workflow.
    • Fig. S3. FSC plots and 3D reconstruction details.
    • Fig. S4. Features of the map and the atomic model of the protein.
    • Fig. S5. Disease-linked mutations in PTCH1 are present within the SSD and SSDL regions of the protein.
    • Fig. S6. Mutations designed to disrupt sterol binding sites in PTCH1 fail to suppress hedgehog pathway activation.
    • Table S1. Cryo-EM data collection, single-particle analysis, and model building statistics.

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). The density elements corresponding to bound sterols in the PTCH1 TM-focused map.

    Files in this Data Supplement:

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