Jinseo Park^1,19, Ziao Fu^2,19, Aurel Frangaj^1,19, Jonathan Liu^1,19, Lidia Mosyak^1,19, Tong Shen^3,19, Vesna N. Slavkovich⁴, Kimberly M. Ray¹, Jaume Taura⁵, Baohua Cao¹, Yong Geng^1,6, Hao Zuo¹, Yongjun Kou⁶, Robert Grassucci², Shaoxia Chen⁷, Zheng Liu², Xin Lin^8,9, Justin P. Williams¹⁰, William J. Rice¹¹, Edward T. Eng¹¹, Rick K. Huang¹², Rajesh K. Soni¹³, Brian Kloss¹⁴, Zhiheng Yu¹², Jonathan A. Javitch^1,8,9,10, Wayne A. Hendrickson^2,10,14, Paul A. Slesinger⁵, Matthias Quick^8,9, Joseph Graziano⁴, Hongtao Yu¹⁵, Oliver Fiehn³, Oliver B. Clarke^10,16,*, Joachim Frank^2,17,* & Qing R. Fan^1,18,*

¹Department of Pharmacology, Columbia University, New York, NY, USA. ²Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA. ³NIH West Coast Metabolomics Center, University of California Davis, Davis, CA, USA. ⁴Department of Environmental Health Sciences, Columbia University, New York, NY, USA. ⁵Nash Family Department of Neuroscience, Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA. ⁶Key Laboratory of Receptor Research, Center for Structure and Function of Drug Targets, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China. ⁷MRC Laboratory of Molecular Biology, Cambridge, UK. ⁸Department of Psychiatry, Columbia University, New York, NY, USA. ⁹Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, NY, USA. ¹⁰Department of Physiology and Cellular Biophysics, Columbia University, New York, NY, USA. ¹¹National Resource for Automated Molecular Microscopy, Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA. ¹²Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA. ¹³Proteomics Shared Resource, Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY, USA. ¹⁴Center on Membrane Protein Production and Analysis, New York Structural Biology Center, New York, NY, USA. ¹⁵Howard Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, USA. ¹⁶Department of Anesthesiology and the Irving Institute for Clinical and Translational Research, Columbia University, New York, NY, USA. ¹⁷Department of Biological Sciences, Columbia University, New York, NY, USA. ¹⁸Department of Pathology and Cell Biology, Columbia University, New York, NY, USA. ¹⁹These authors contributed equally: Jinseo Park, Ziao Fu, Aurel Frangaj, Jonathan Liu, Lidia Mosyak, Tong Shen.

^*Corresponding authors

The human GABAB receptor—a member of the class C family of G-protein-coupled receptors (GPCRs)—mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2,3,4,5,6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9,10,11,12,13,14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique ‘intersubunit latch’ within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.