Structural analysis of G-protein-coupled receptors (GPCRs) for hormones and neurotransmitters has been hindered by their low natural abundance, inherent structural flexibility, and instability in detergent solutions. Here we report a structure of the human beta2 adrenoceptor (beta2AR), which was crystallized in a lipid environment when bound to an inverse agonist and in complex with a Fab that binds to the third intracellular loop. Diffraction data were obtained by high-brilliance microcrystallography and the structure determined at 3.4 A/3.7 A resolution. The cytoplasmic ends of the beta2AR transmembrane segments and the connecting loops are well resolved, whereas the extracellular regions of the beta2AR are not seen. The beta2AR structure differs from rhodopsin in having weaker interactions between the cytoplasmic ends of transmembrane (TM)3 and TM6, involving the conserved E/DRY sequences. These differences may be responsible for the relatively high basal activity and structural instability of the beta2AR, and contribute to the challenges in obtaining diffraction-quality crystals of non-rhodopsin GPCRs.

G-protein-coupled receptors (GPCRs) are physiologically important membrane proteins that sense signalling molecules such as hormones and neurotransmitters, and are the targets of several prescribed drugs. Recent exciting developments are providing unprecedented insights into the structure and function of several medically important GPCRs. Here, through a systematic analysis of high-resolution GPCR structures, we uncover a conserved network of non-covalent contacts that defines the GPCR fold. Furthermore, our comparative analysis reveals characteristic features of ligand binding and conformational changes during receptor activation. A holistic understanding that integrates molecular and systems biology of GPCRs holds promise for new therapeutics and personalized medicine.

SummaryG protein-coupled receptors play a major role in transmembrane signalling in higher organisms and many are important drug targets. We report the 2.7 Å resolution crystal structure of a β 1 -adrenergic receptor in complex with the high-affinity antagonist cyanopindolol. The modified turkey receptor had been selected to be in its antagonist conformation and its thermostability improved by earlier limited mutagenesis. The ligand-binding pocket comprises 15 side chains from amino acid residues in 4 transmembrane α-helices and extracellular loop 2. This loop defines the entrance of the ligand-binding pocket and is stabilised by two disulphide bonds and a sodium ion. Cyanopindolol binding to the β 1 -adrenergic receptor and carazolol binding to the β 2 -adrenergic receptor involve similar interactions. A short well-defined helix in cytoplasmic loop 2, not observed in either rhodopsin or the β 2 -adrenergic receptor, directly interacts via a tyrosine with the highly conserved DRY motif at the end of helix 3 that is essential for receptor activation.G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins that are prevalent in eukaryotes from yeast to man, and which function as key intermediaries in the transduction of signals from outside to inside the cell1. Activating molecules (agonists), such as hormones and neurotransmitters, bind to GPCRs from the extracellular side of the cell membrane and induce a large conformational change which propagates to the cytoplasmic surface2,3, resulting in activation of G proteins and a consequent change in the level of intracellular messengers such as cAMP, Ca 2+ or signalling lipids. There are over 800 different human GPCRs4, all sharing the characteristic arrangement of 7 transmembrane α-helices with the polypeptide N-terminus on the extracellular side of the plasma membrane5. Analysis of their primary amino acid sequences has resulted in the definition of a number of families6, the largest of which, family A, includes the archetypal GPCR, rhodopsin. The three human β-adrenergic receptor (βAR) subtypes, β 1 , β 2 and β 3 , belong to family A and share 51% sequence identity between Trp 1.31 -Asp 5.73 and Glu 6.30 -Cys H8-Cterm i.e. excluding the N-and C-termini and most of cytoplasmic loop 3 ( Supplementary Fig 1; superscripts refer to Ballesteros-Weinstein numbering7). Drugs that * Joint corresponding authors MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK cgt@mrc-lmb.cam.ac.ukgfx@mrc-lmb.cam.ac.uk Telephone +44-(0)1223-402338 +44-(0)1223-402328 Fax +44-(0)1223-213556 . Author contributions. TW devised and carried out receptor expression, purification, crystallisation and cryo-cooling of the crystals. Receptor stabilisation and baculovirus expression were performed by MJSV; both authors were also involved in data collection and preliminary crystallographic analyses of the crystals. PE helped with the crystal cryo-cooling strategy and in diffraction data collection. JGB performed the functional cAMP and reporter gene assays...

No abstract