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Nanobody-enabled monitoring of kappa opioid receptor states.
- Source :
-
Nature communications [Nat Commun] 2020 Mar 02; Vol. 11 (1), pp. 1145. Date of Electronic Publication: 2020 Mar 02. - Publication Year :
- 2020
-
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.
- Subjects :
- 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 genetics
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Single-Domain Antibodies metabolism
Tetrahydroisoquinolines chemistry
Tetrahydroisoquinolines pharmacology
Receptors, Opioid, kappa chemistry
Receptors, Opioid, kappa metabolism
Single-Domain Antibodies chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 2041-1723
- Volume :
- 11
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Nature communications
- Publication Type :
- Academic Journal
- Accession number :
- 32123179
- Full Text :
- https://doi.org/10.1038/s41467-020-14889-7