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Nanobody-enabled monitoring of kappa opioid receptor states

Author

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  • Tao Che

    (University of North Carolina at Chapel Hill School of Medicine)

  • Justin English

    (University of North Carolina at Chapel Hill School of Medicine)

  • Brian E. Krumm

    (University of North Carolina at Chapel Hill School of Medicine)

  • Kuglae Kim

    (University of North Carolina at Chapel Hill School of Medicine)

  • Els Pardon

    (Vrije Universiteit Brussel (VUB)
    VIB-VUB Center for Structural Biology, VIB)

  • Reid H. J. Olsen

    (University of North Carolina at Chapel Hill School of Medicine)

  • Sheng Wang

    (University of North Carolina at Chapel Hill School of Medicine
    University of Chinese Academy of Sciences)

  • Shicheng Zhang

    (University of North Carolina at Chapel Hill School of Medicine)

  • Jeffrey F. Diberto

    (University of North Carolina at Chapel Hill School of Medicine)

  • Noah Sciaky

    (University of North Carolina at Chapel Hill School of Medicine)

  • F. Ivy Carroll

    (Research Triangle Institute, Research Triangle Park)

  • Jan Steyaert

    (Vrije Universiteit Brussel (VUB)
    VIB-VUB Center for Structural Biology, VIB)

  • Daniel Wacker

    (University of North Carolina at Chapel Hill School of Medicine
    Department of Pharmacological Sciences and Department of Neuroscience, Icahn School of Medicine at Mount Sinai)

  • Bryan L. Roth

    (University of North Carolina at Chapel Hill School of Medicine
    University of North Carolina at Chapel Hill
    University of North Carolina at Chapel Hill School of Medicine)

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.

Suggested Citation

  • 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 , 2020. "Nanobody-enabled monitoring of kappa opioid receptor states," Nature Communications, Nature, vol. 11(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-14889-7
    DOI: 10.1038/s41467-020-14889-7
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    Cited by:

    1. Yong-Seok Kim & Jun-Hee Yeon & Woori Ko & Byung-Chang Suh, 2023. "Two-step structural changes in M3 muscarinic receptor activation rely on the coupled Gq protein cycle," Nature Communications, Nature, vol. 14(1), pages 1-18, December.
    2. Amal El Daibani & Joseph M. Paggi & Kuglae Kim & Yianni D. Laloudakis & Petr Popov & Sarah M. Bernhard & Brian E. Krumm & Reid H. J. Olsen & Jeffrey Diberto & F. Ivy Carroll & Vsevolod Katritch & Bern, 2023. "Molecular mechanism of biased signaling at the kappa opioid receptor," Nature Communications, Nature, vol. 14(1), pages 1-13, December.
    3. Xiao Teng & Sijia Chen & Yingying Nie & Peng Xiao & Xiao Yu & Zhenhua Shao & Sanduo Zheng, 2022. "Ligand recognition and biased agonism of the D1 dopamine receptor," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    4. Yosuke Toyoda & Angqi Zhu & Fang Kong & Sisi Shan & Jiawei Zhao & Nan Wang & Xiaoou Sun & Linqi Zhang & Chuangye Yan & Brian K. Kobilka & Xiangyu Liu, 2023. "Structural basis of α1A-adrenergic receptor activation and recognition by an extracellular nanobody," Nature Communications, Nature, vol. 14(1), pages 1-13, December.

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