Research Article

Structural basis for the recognition of Sonic Hedgehog by human Patched1

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Science  10 Aug 2018:
Vol. 361, Issue 6402, eaas8935
DOI: 10.1126/science.aas8935

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The first step in Hedgehog signaling

The Hedgehog (Hh) signaling pathway is important in embryogenesis; overactivation is associated with cancer. Central to the pathway is the membrane receptor Patched 1 (Ptch1), which indirectly inhibits a G protein–coupled receptor called Smoothened. This inhibition is relieved when Ptch1 binds the secreted protein Hh. Gong et al. report the cryo–electron microscopy structures of human Ptch1 alone and in complex with its Hh ligand at 3.9 and 3.6 Å, respectively. Both structures include two steroid-shaped densities, and mutational analysis indicates that the interaction between Ptch1 and Hh is steroid-dependent.

Science, this issue p. eaas8935

Structured Abstract

INTRODUCTION

The Hedgehog (Hh) pathway is critical for embryogenesis and tissue regeneration. Hh signaling is activated by binding of the secreted and lipid-modified protein Hh to the membrane receptor Patched (Ptch). In the absence of Hh, Ptch suppresses the downstream G protein–coupled receptor Smoothened (Smo) via an unknown and indirect mechanism. Binding of Hh to Ptch relieves the inhibition on Smo and turns on the signaling events that lead to the transcriptional activation of the Hh pathway. Aberrations of Hh signaling are associated with birth defects or tumorigenesis. Despite rigorous investigations, the molecular basis for the interplay among Hh, Ptch, and Smo remains unclear, and the structural basis for the recognition between Ptch and Hh is yet to be elucidated.

The 1447-residue human Ptch1 protein is predicted to contain 12 transmembrane segments (TMs) and to share structural similarity with the bacterial resistance-nodulation-division (RND) family transporters. TMs 2 to 6 of Ptch1 constitute the sterol-sensing domain (SSD), which has been found in several proteins involved in sterol transport and metabolism. The molecular mechanism for potential sterol binding or transport activity of these SSD-containing proteins remains elusive.

RATIONALE

To obtain a sample suitable for structural study, we generated several constructs of human Ptch1 based on sequence conservation and functional characterizations. Eventually, the truncated human Ptch1 containing residues 1 to 1305, which was transiently expressed in human embryonic kidney (HEK) 293F cells, exhibited a sufficient expression level and good solution behavior after affinity and size exclusion chromatography purification. Both oligomeric and monomeric states of Ptch1 were observed. The monomeric form was used for single-particle cryo–electron microscopy (cryo-EM) analysis because of its excellent behavior under cryo conditions.

Among the three mammalian Hh homologs Sonic (Shh), Desert (Dhh), and Indian (Ihh), Shh has been the prototype for functional and mechanistic investigations. The N-terminal domain of human Sonic Hh (ShhN, residues 24 to 197) expressed and purified in Escherichia coli was able to form a stable complex with the detergent-solubilized Ptch1 protein in the presence of cholesteryl hemisuccinate (CHS).

RESULTS

The cryo-EM structures of human Ptch1 alone and in complex with ShhN were determined at overall resolutions of 3.9 Å and 3.6 Å, respectively. Two interacting extracellular domains, ECD1 and ECD2, and 12 TMs are resolved for Ptch1. ECD1 and ECD2 move toward each other upon arrival of ShhN and together constitute the docking site for ShhN. Detailed recognition between ShhN and Ptch1 was analyzed and biochemically confirmed.

Two elongated densities, both consistent with CHS, are observed in Ptch1 with or without ShhN, one in a pocket enclosed by the ECD domains and the other in a membrane-facing cavity of the SSD. Structure-guided biochemical analyses revealed steroid-dependent interaction between ShhN and Ptch1. The structure of a steroid binding–deficient Ptch1 mutant displays pronounced conformational rearrangements relative to the wild-type protein.

CONCLUSION

The structures of human Ptch1 and its complex with ShhN reveal the molecular basis for the recognition between Ptch1 and ShhN. The identification of two steroid-binding sites in Ptch1 establishes an important framework for future investigations of Hh signaling and provides critical insight into sterol perception by SSD-containing proteins.

Structural basis for sterol perception by the sterol-sensing domain (SSD) and steroid-dependent binding between Ptch1 and ShhN.

(A) Structural comparison of Ptch1 alone (silver) and in complex with ShhN (domain-colored). (B) Two CHS binding sites on Ptch1, one enclosed by extracellular domains [the extracellular steroid binding site (ESBS)] and one on the SSD. (C) Potential sterol transfer between the two binding sites. (D) Functional implications of the structures.

Abstract

The Hedgehog (Hh) pathway involved in development and regeneration is activated by the extracellular binding of Hh to the membrane receptor Patched (Ptch). We report the structures of human Ptch1 alone and in complex with the N-terminal domain of human Sonic hedgehog (ShhN) at resolutions of 3.9 and 3.6 angstroms, respectively, as determined by cryo–electron microscopy. Ptch1 comprises two interacting extracellular domains, ECD1 and ECD2, and 12 transmembrane segments (TMs), with TMs 2 to 6 constituting the sterol-sensing domain (SSD). Two steroid-shaped densities are resolved in both structures, one enclosed by ECD1/2 and the other in the membrane-facing cavity of the SSD. Structure-guided mutational analysis shows that interaction between ShhN and Ptch1 is steroid-dependent. The structure of a steroid binding–deficient Ptch1 mutant displays pronounced conformational rearrangements.

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