Nanobody-enabled monitoring of kappa opioid receptor states

Tao Che, Justin English, Brian E. Krumm, Kuglae Kim, Els Pardon, Reid H.J. Olsen, Sheng Wang, Shicheng Zhang, Jeffrey F. Diberto, Noah Sciaky, F. Ivy Carroll, Jan Steyaert, Daniel Wacker, Bryan L. Roth

Research output: Contribution to journalArticlepeer-review

43 Citations (Scopus)

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.

Original languageEnglish
Article number1145
JournalNature communications
Volume11
Issue number1
DOIs
Publication statusPublished - 2020 Dec 1

Bibliographical note

Funding Information:
This work was supported by NIH grants (PO1DA0357634; R37DA045657, and RO1MH112205), the NIMH Psychoactive Drug Screening Program Contract, and the Michael Hooker Distinguished Chair of Pharmacology (to B.L.R.). We thank INSTRUCT, part of the European Strategy Forum on Research Infrastructures (ESFRI), and the Research Foundation—Flanders (FWO) for their support to the nanobody discovery and thank Nele Buys and Katleen Willibal for the technical assistance. R.H.J. Olsen was also supported by grant F31-NS093917. We thank Michael S. Placzek for providing LY2459989. We also thank Roshanak Irannejad for providing the GalT-RFP construct. We gratefully acknowledge M.J. Miley and the UNC Macromolecular Crystallization Core for advice and use of their equipment for crystal harvesting and transport, which is supported by the National Cancer Institute (award number P30CA016086). We thank J. Smith and R. Fischetti and the staff of GM/CA@APS, which has been funded with Federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility, operated for the DOE Office of Science by Argonne National Laboratory (contract no. DE-AC02-06CH11357).

Publisher Copyright:
© 2020, The Author(s).

All Science Journal Classification (ASJC) codes

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physics and Astronomy(all)

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