Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm

Hongwu Qian,Nieng Yan,Miaohui Hu,Xin Gong,Pingping Cao,Shuai Gao

doi:10.1038/s41467-019-10234-9

Abstract

The Hedgehog (Hh) pathway controls embryonic development and postnatal tissue maintenance and regeneration. Inhibition of Hh receptor Patched (Ptch) by the Hh ligands relieves suppression of signaling cascades. Here, we report the cryo-EM structure of tetrameric Ptch1 in complex with the palmitoylated N-terminal signaling domain of human Sonic hedgehog (ShhNp) at a 4:2 stoichiometric ratio. The structure shows that four Ptch1 protomers are organized as a loose dimer of dimers. Each dimer binds to one ShhNp through two distinct inhibitory interfaces, one mainly through the N-terminal peptide and the palmitoyl moiety of ShhNp and the other through the Ca2+-mediated interface on ShhNp. Map comparison reveals that the cholesteryl moiety of native ShhN occupies a recently identified extracellular steroid binding pocket in Ptch1. Our structure elucidates the tetrameric assembly of Ptch1 and suggests an asymmetric mode of action of the Hh ligands for inhibiting the potential cholesterol transport activity of Ptch1.

Highlights

The Hedgehog (Hh) pathway controls embryonic development and postnatal tissue maintenance and regeneration
The major species of this Ptch[1] construct existed in an oligomeric form upon size-exclusion chromatography (SEC) (Supplementary Fig. 1b), the minor monomeric form was used for cryo-EM analysis in our previous study
The cryo-sample became amenable for cryo-EM analysis when glycol-diosgenin (GDN, Anatrace) was used for protein extraction and purification

Summary

Introduction

The Hedgehog (Hh) pathway controls embryonic development and postnatal tissue maintenance and regeneration. Our structure elucidates the tetrameric assembly of Ptch[1] and suggests an asymmetric mode of action of the Hh ligands for inhibiting the potential cholesterol transport activity of Ptch[1]. Binding of the processed Hh ligand to the surface receptor Patched (Ptch) relieves suppression of the downstream G-protein coupled receptor, Smoothened (Smo). This subsequently activates the Hh signaling cascade that controls embryogenesis and tissue homeostasis[4,5,6]. Ptch shares sequence homology with the prokaryotic resistancenodulation-division (RND) family transporters, exemplified by the bacterial proton-driven multidrug resistance exporter AcrB16 This phylogenetic evidence led to a model suggesting that Ptch may act as a transporter for the ligands, antagonists or agonists, of Smo

Methods

Results

Conclusion