GPCR engineering yields high-resolution structural insights into beta2-adrenergic receptor function.
The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.
Rosenbaum
Daniel M
DM
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA.
Cherezov
Vadim
V
Hanson
Michael A
MA
Rasmussen
Søren G F
SG
Thian
Foon Sun
FS
Kobilka
Tong Sun
TS
Choi
Hee-Jung
HJ
Yao
Xiao-Jie
XJ
Weis
William I
WI
Stevens
Raymond C
RC
Kobilka
Brian K
BK
D000318:Adrenergic beta-Agonists / Q000737:chemistry / Q000378:metabolism; D000319:Adrenergic beta-Antagonists / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D054314:Drug Inverse Agonism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D011412:Propanolamines / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D000318:Adrenergic beta-Agonists; D000319:Adrenergic beta-Antagonists; D007140:Immunoglobulin Fab Fragments; D008024:Ligands; D011412:Propanolamines; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; C014382:carazolol; D009113:Muramidase