Nanobody-enabled monitoring of kappa opioid receptor states

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 con...

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Published in:	Nature communications 2020-03, Vol.11 (1), p.1145-1145, Article 1145
Main Authors:	Che, Tao, English, Justin, Krumm, Brian E, Kim, Kuglae, Pardon, Els, Olsen, Reid H J, Wang, Sheng, Zhang, Shicheng, Diberto, Jeffrey F, Sciaky, Noah, Carroll, F Ivy, Steyaert, Jan, Wacker, Daniel, Roth, Bryan L
Format:	Article
Language:	eng
Subjects:	60 APPLIED LIFE SCIENCES Allosteric properties Allosteric Site Binding Sites Biosensing Techniques Crystallography Crystallography, X-Ray Cyclic AMP - metabolism Dynorphins - chemistry Dynorphins - pharmacology G protein-coupled receptors HEK293 Cells Humans Ligands Luminescent Measurements - methods Narcotics Opioid receptors (type kappa) Piperazines - chemistry Piperazines - pharmacology Piperidines - chemistry Piperidines - pharmacology Protein Conformation Pyrrolidines - chemistry Pyrrolidines - pharmacology Real time Receptors 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 Time dependence X-ray crystallography
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Summary:	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.
ISSN:	2041-1723 2041-1723