title: Structural basis of Smoothened regulation by its extracellular domains.
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.
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.
authors.forename: Eamon F X
authors.initials: EFX
authors.lastname: Byrne
authors.affiliation: Departments of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Ria
authors.initials: R
authors.lastname: Sircar
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Miller
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford, UK.
authors.forename: George
authors.initials: G
authors.lastname: Hedger
authors.affiliation: Departments of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Luchetti
authors.affiliation: Departments of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Sigrid
authors.initials: S
authors.lastname: Nachtergaele
authors.affiliation: Diamond Light Source Ltd, Harwell Science &Innovation Campus, Didcot, UK.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Tully
authors.affiliation: Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri, United States of America.
authors.forename: Laurel
authors.initials: L
authors.lastname: Mydock-McGrane
authors.affiliation: Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri, United States of America.
authors.forename: Douglas F
authors.initials: DF
authors.lastname: Covey
authors.affiliation: Diamond Light Source Ltd, Harwell Science &Innovation Campus, Didcot, UK.
authors.forename: Robert P
authors.initials: RP
authors.lastname: Rambo
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford, UK.
authors.forename: Mark S P
authors.initials: MSP
authors.lastname: Sansom
authors.affiliation: Department of Biochemistry, University of Oxford, Oxford, UK.
authors.forename: Simon
authors.initials: S
authors.lastname: Newstead
authors.affiliation: Departments of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Rajat
authors.initials: R
authors.lastname: Rohatgi
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Siebold
mesh_terms: D000813:Anilides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics; D002784:Cholesterol / Q000378:metabolism / Q000494:pharmacology; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism*; D053823:Hedgehog Proteins / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D011485:Protein Binding / Q000235:genetics; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D000072086:Smoothened Receptor
chemical_list: D000813:Anilides; D000970:Antineoplastic Agents; D053823:Hedgehog Proteins; C538724:HhAntag691; D008024:Ligands; D011725:Pyridines; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor; D002784:Cholesterol; D003545:Cysteine