Journal Article

Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling

Nachtergaele, Sigrid, Whalen, Daniel M, Krishnan, Kathiresan, Ingham, Philip W, Mydock, Laurel K, Siebold, Christian, Malinauskas, Tomas, Covey, Douglas F, Rohatgi, Rajat, Zhao, Zhonghua

2013

Overview

The Hedgehog (Hh) signal is transduced across the membrane by the heptahelical protein Smoothened (Smo), a developmental regulator, oncoprotein and drug target in oncology. We present the 2.3 Å crystal structure of the extracellular cysteine rich domain (CRD) of vertebrate Smo and show that it binds to oxysterols, endogenous lipids that activate Hh signaling. The oxysterol-binding groove in the Smo CRD is analogous to that used by Frizzled 8 to bind to the palmitoleyl group of Wnt ligands and to similar pockets used by other Frizzled-like CRDs to bind hydrophobic ligands. The CRD is required for signaling in response to native Hh ligands, showing that it is an important regulatory module for Smo activation. Indeed, targeting of the Smo CRD by oxysterol-inspired small molecules can block signaling by all known classes of Hh activators and by clinically relevant Smo mutants. Just over 30 years ago, researchers identified a new signaling molecule with an important role in the development of fruit flies. Embryos lacking this molecule were thought to resemble a hedgehog, eventually leading to this cell–cell communication system being designated the “Hedgehog” pathway. This pathway has subsequently been shown to be involved in the development of many other animals, as well as in the repair of damaged tissues in adult organisms. Abnormal Hedgehog signaling has also been implicated in both human birth defects and in cancers of the skin and the brain. Many such tumors are driven by the unrestrained activation of a membrane-bound protein called Smoothened, which has led to the development and clinical use of small molecules that prevent Hedgehog from activating Smoothened. The existing anti-tumor drugs all bind to the same region of the Smoothened receptor, namely the part that sits within the cell membrane. A second group of molecules, known as oxysterols, can activate Smoothened, but exactly how they do this has been unclear. Now, Nachtergaele et al. have shown that oxysterols bind to a region of the Smoothened receptor that lies outside the cell, and that is rich in the amino acid cysteine. By solving the crystal structure of this part of the receptor from zebrafish, Nachtergaele et al. were able to map the oxysterol binding site at high resolution. This revealed strong similarities between this binding site and those in related receptors belonging to the Wnt signaling pathway. Deleting the cysteine-rich domain significantly impaired Hedgehog signaling, as did a new class of small molecule inhibitors designed specifically to target the oxysterol binding site. In addition to providing new insights into the structure and function of the Smoothened receptor, the work of Nachtergaele et al. opens up possibilities for novel therapeutic agents that could be used in the treatment of cancers caused by abnormal Hedgehog signaling.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Animals / Binding Sites / Biochemistry / Biology / Biophysics and Structural Biology / Chromatography / Crystal structure / Crystallography, X-Ray / cysteine rich domain / Embryo, Nonmammalian / Escherichia coli - genetics / Escherichia coli - metabolism / Frizzled protein / Gene Expression Regulation, Developmental / Genes / Hedgehog protein / Hedgehog Proteins - chemistry / Hedgehog Proteins - genetics / Hedgehog Proteins - metabolism / Hedgehog signaling / Hydrophobicity / Ligands / Lipids / Medicine / Membrane proteins / Mice / Models, Molecular / oxysterol / Protein Binding / Protein Structure, Secondary / Protein Structure, Tertiary / Proteins / Receptors, G-Protein-Coupled - antagonists & inhibitors / Receptors, G-Protein-Coupled - chemistry / Receptors, G-Protein-Coupled - genetics / Receptors, G-Protein-Coupled - metabolism / Recombinant Proteins - chemistry / Recombinant Proteins - genetics / Recombinant Proteins - metabolism / Signal Transduction / smoothened / Smoothened Receptor / Sterols - chemistry / Structure-Activity Relationship / Structure-function relationships / Wnt protein / Zebrafish - genetics / Zebrafish - growth & development / Zebrafish - metabolism / Zebrafish Proteins - antagonists & inhibitors / Zebrafish Proteins - chemistry / Zebrafish Proteins - genetics / Zebrafish Proteins - metabolism