Structural basis of Smoothened regulation by its extracellular domains

Nature. 2016 Jul 28;535(7613):517-522. doi: 10.1038/nature18934. Epub 2016 Jul 20.

Abstract

Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Anilides / chemistry
  • Anilides / metabolism
  • Anilides / pharmacology
  • Antineoplastic Agents / metabolism
  • Antineoplastic Agents / pharmacology
  • Binding Sites / genetics
  • Cholesterol / metabolism
  • Cholesterol / pharmacology
  • Crystallography, X-Ray
  • Cysteine / chemistry
  • Cysteine / genetics
  • Cysteine / metabolism
  • Extracellular Space / metabolism*
  • Hedgehog Proteins / metabolism
  • Humans
  • Ligands
  • Models, Molecular
  • Protein Binding / genetics
  • Protein Stability / drug effects
  • Protein Structure, Tertiary / drug effects
  • Protein Structure, Tertiary / genetics
  • Pyridines / chemistry
  • Pyridines / metabolism
  • Pyridines / pharmacology
  • Receptors, G-Protein-Coupled / antagonists & inhibitors
  • Receptors, G-Protein-Coupled / chemistry*
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism*
  • Signal Transduction / drug effects
  • Smoothened Receptor

Substances

  • Anilides
  • Antineoplastic Agents
  • Hedgehog Proteins
  • HhAntag691
  • Ligands
  • Pyridines
  • Receptors, G-Protein-Coupled
  • SMO protein, human
  • Smoothened Receptor
  • Cholesterol
  • Cysteine