Research Article

Two Patched molecules engage distinct sites on Hedgehog yielding a signaling-competent complex

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Science  05 Oct 2018:
Vol. 362, Issue 6410, eaas8843
DOI: 10.1126/science.aas8843

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It takes two to signal

The Hedgehog (HH) signaling pathway is important in development, and excessive HH signaling is associated with cancer. Signaling occurs through the G protein–coupled receptor Smoothened. The pathway is repressed by the membrane receptor Patched-1 (PTCH1), and this inhibition is relieved when PTCH1 binds the secreted protein HH. Two recent papers have described structures of HH bound to PTCH1, but surprisingly, each described a different binding epitope on HH. Qi et al. present a cryo–electron microscopy structure that explains this apparent contradiction by showing that a single HH protein uses both of these interfaces to engage two PTCH1 receptors (see the Perspective by Sommer and Lemmon). Functional assays suggest that both interfaces must be bound for efficient signaling.

Science, this issue p. eaas8843; see also p. 26

Structured Abstract


Appropriate Hedgehog (HH) signaling is important for human health; excessive HH signaling leads to various cancers, whereas insufficient HH signaling results in birth defects. The mature N-terminal domain of the HH protein with N-terminal palmitate and C-terminal cholesterol modifications (referred to as “native HH-N” here) binds to its receptor, Patched-1 (PTCH1), to release its repression of the HH pathway, presumably by regulating the transport of a ligand that regulates the downstream oncoprotein Smoothened. The palmitate moiety of HH-N is essential for HH signaling; however, the mechanism of how the palmitate moiety of HH induces the signaling is not clear. The means by which HH protein recognizes PTCH1 is also controversial: A recent 1:1 PTCH1–HH-N complex structure visualized a palmitate-mediated binding site on HH-N, which was inconsistent with previous studies that implied a distinct, Ca2+-mediated interface for PTCH1 and HH co-receptors.


Cell biological studies indicated that mutations on either the palmitate-dominated interface or the Ca2+-mediated interface of HH-N eliminated signaling, indicating that both HH-N interfaces are necessary for HH to inhibit PTCH1 activity. We managed to assemble a 2:1 PTCH1–HH-N complex using a biologically functional PTCH1 variant (PTCH1*) and native Sonic HH-N (SHH-N) in the presence of 1 mM CaCl2 (a physiological extracellular calcium concentration) for structural investigation.


The cryo–electron microscopy (cryo-EM) structure of native SHH-N in complex with PTCH1* at 3.5-Å resolution demonstrates that one SHH-N molecule concomitantly engages both epitopes and binds two PTCH1 receptors (PTCH1-A and PTCH1-B) in an asymmetric manner. PTCH1-A and PTCH1-B exhibit several conformational differences and bind distinct portions of SHH-N. Structural comparisons suggest that the Ca2+-mediated interface of SHH-N physiologically binds different HH binders, including PTCH1, HH co-receptors, and the SHH-N antagonist 5E1 antibody, to regulate HH signaling. Functional assays using PTCH1 or SHH-N mutants that disrupt the individual interfaces illustrate that simultaneous engagement of both interfaces between SHH-N and PTCH1 is essential for maximal signaling in cells.

Calculations using MOLE reveal a tunnel, ~150 Å in length with a minimum radius of 4 Å, that stretches through PTCH1-B but not PTCH1-A, which interacts with the palmitate of SHH-N. The path of this PTCH1-B tunnel includes the extracellular domain I (ECD-I), the cavity of the ECDs that accommodates the palmitate, and the transmembrane region. We introduced two point mutations (L427R and F1017E) on full-length human PTCH1 to block this tunnel by a ~3-Å salt bridge instead of by palmitate insertion. Cell biological assays show that unlike wild-type PTCH1, this variant was not able to repress the HH pathway; this finding supports our hypothesis that the palmitate can abolish the activity of PTCH1 by blocking the cavity of the ECDs.


Our structure of a signaling-competent complex consisting of SHH-N and an asymmetric arrangement of two PTCH1* molecules reconciles previous disparate findings of how HH-N interacts with PTCH1. The structural analysis reveals a tunnel in the PTCH1 molecule that can be blocked using mutations in the ECDs that mimic the palmitate insertion, thus abolishing HH pathway repression by PTCH1. Thus, our work illuminates how two sites on SHH-N serve to unite two PTCH1 receptors, thereby initiating a full HH signal.

Cryo-EM structure of 2:1 human PTCH1*–SHH-N signaling-competent complex.

PTCH1-A, PTCH1-B, and SHH-N are colored yellow, light blue, and cyan, respectively, and are viewed from the side of the membrane. Palmitate (PLM) is shown as magenta sticks, calcium as green spheres. Cholesterol in the cell membrane is shown as ball-and-stick models colored yellow and red.


Aberrant Hedgehog (HH) signaling leads to various types of cancer and birth defects. N-terminally palmitoylated HH initiates signaling by binding its receptor Patched-1 (PTCH1). A recent 1:1 PTCH1-HH complex structure visualized a palmitate-mediated binding site on HH, which was inconsistent with previous studies that implied a distinct, calcium-mediated binding site for PTCH1 and HH co-receptors. Our 3.5-angstrom resolution cryo–electron microscopy structure of native Sonic Hedgehog (SHH-N) in complex with PTCH1 at a physiological calcium concentration reconciles these disparate findings and demonstrates that one SHH-N molecule engages both epitopes to bind two PTCH1 receptors in an asymmetric manner. Functional assays using PTCH1 or SHH-N mutants that disrupt the individual interfaces illustrate that simultaneous engagement of both interfaces is required for efficient signaling in cells.

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