Nanobody-enabled monitoring of kappa opioid receptor states

Nat Commun. 2020 Mar 2;11(1):1145. doi: 10.1038/s41467-020-14889-7.

Abstract

Recent studies show that GPCRs rapidly interconvert between multiple states although our ability to interrogate, monitor and visualize them is limited by a relative lack of suitable tools. We previously reported two nanobodies (Nb39 and Nb6) that stabilize distinct ligand- and efficacy-delimited conformations of the kappa opioid receptor. Here, we demonstrate via X-ray crystallography a nanobody-targeted allosteric binding site by which Nb6 stabilizes a ligand-dependent inactive state. As Nb39 stabilizes an active-like state, we show how these two state-dependent nanobodies can provide real-time reporting of ligand stabilized states in cells in situ. Significantly, we demonstrate that chimeric GPCRs can be created with engineered nanobody binding sites to report ligand-stabilized states. Our results provide both insights regarding potential mechanisms for allosterically modulating KOR with nanobodies and a tool for reporting the real-time, in situ dynamic range of GPCR activity.

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

  • Allosteric Site
  • Binding Sites
  • Biosensing Techniques
  • Crystallography, X-Ray
  • Cyclic AMP / metabolism
  • Dynorphins / chemistry
  • Dynorphins / pharmacology
  • HEK293 Cells
  • Humans
  • Luminescent Measurements / methods
  • Piperazines / chemistry
  • Piperazines / pharmacology
  • Piperidines / chemistry
  • Piperidines / pharmacology
  • Protein Conformation
  • Pyrrolidines / chemistry
  • Pyrrolidines / pharmacology
  • Receptors, G-Protein-Coupled / chemistry
  • Receptors, G-Protein-Coupled / metabolism
  • Receptors, Opioid, kappa / agonists
  • Receptors, Opioid, kappa / chemistry*
  • Receptors, Opioid, kappa / genetics
  • Receptors, Opioid, kappa / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Single-Domain Antibodies / chemistry*
  • Single-Domain Antibodies / metabolism
  • Tetrahydroisoquinolines / chemistry
  • Tetrahydroisoquinolines / pharmacology

Substances

  • 7-hydroxy-N-(1-((4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl)methyl)-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide
  • Piperazines
  • Piperidines
  • Pyrrolidines
  • Receptors, G-Protein-Coupled
  • Receptors, Opioid, kappa
  • Recombinant Proteins
  • Single-Domain Antibodies
  • Tetrahydroisoquinolines
  • GR 89696
  • Dynorphins
  • Cyclic AMP