Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody

Nature. 2013 Oct 24;502(7472):575-579. doi: 10.1038/nature12572. Epub 2013 Sep 22.

Abstract

G-protein-coupled receptors (GPCRs) are integral membrane proteins that have an essential role in human physiology, yet the molecular processes through which they bind to their endogenous agonists and activate effector proteins remain poorly understood. So far, it has not been possible to capture an active-state GPCR bound to its native neurotransmitter. Crystal structures of agonist-bound GPCRs have relied on the use of either exceptionally high-affinity agonists or receptor stabilization by mutagenesis. Many natural agonists such as adrenaline, which activates the β2-adrenoceptor (β2AR), bind with relatively low affinity, and they are often chemically unstable. Using directed evolution, we engineered a high-affinity camelid antibody fragment that stabilizes the active state of the β2AR, and used this to obtain crystal structures of the activated receptor bound to multiple ligands. Here we present structures of the active-state human β2AR bound to three chemically distinct agonists: the ultrahigh-affinity agonist BI167107, the high-affinity catecholamine agonist hydroxybenzyl isoproterenol, and the low-affinity endogenous agonist adrenaline. The crystal structures reveal a highly conserved overall ligand recognition and activation mode despite diverse ligand chemical structures and affinities that range from 100 nM to ∼80 pM. Overall, the adrenaline-bound receptor structure is similar to the others, but it has substantial rearrangements in extracellular loop three and the extracellular tip of transmembrane helix 6. These structures also reveal a water-mediated hydrogen bond between two conserved tyrosines, which appears to stabilize the active state of the β2AR and related GPCRs.

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

  • Adrenergic beta-2 Receptor Agonists / pharmacology*
  • Benzoxazines / pharmacology
  • Binding Sites / drug effects
  • Crystallography, X-Ray
  • Directed Molecular Evolution
  • Epinephrine / pharmacology*
  • Humans
  • Hydrogen Bonding / drug effects
  • Isoproterenol / analogs & derivatives
  • Isoproterenol / pharmacology
  • Ligands
  • Models, Molecular
  • Protein Engineering*
  • Protein Stability / drug effects
  • Receptors, Adrenergic, beta-2 / chemistry*
  • Receptors, Adrenergic, beta-2 / drug effects
  • Receptors, Adrenergic, beta-2 / metabolism*
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / pharmacology*
  • Tyrosine / chemistry
  • Tyrosine / metabolism
  • Water / chemistry
  • Water / pharmacology

Substances

  • Adrenergic beta-2 Receptor Agonists
  • BI167107
  • Benzoxazines
  • Ligands
  • Receptors, Adrenergic, beta-2
  • Single-Chain Antibodies
  • Water
  • Tyrosine
  • hydroxybenzylisoproterenol
  • Isoproterenol
  • Epinephrine

Associated data

  • PDB/4LDE
  • PDB/4LDL
  • PDB/4LDO